Molecular Tools for the Detection and Characterization of Bacterial Infections: A Review

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1 Molecular Tools for the Detection and Characterization of Bacterial Infections: A Review Alagarraju Muthukumar, PhD, 1 Nicole L. Zitterkopf, PhD, D(ABMM), 2 Deborah Payne, PhD, DABCC, D(ABMM), FACB 3 ( 1 Clinical Chemistry Research and Development, Abbott Diagnostics, Irving, TX, 2 Clinical Laboratory Sciences, University of Minnesota Rochester, Minneapolis, MN, 3 Molecular Diagnostics Laboratory, UT Southwestern Medical Center, Dallas, TX) DOI: /M6MBU1KGP0FF1C00 Abstract Bacterial infections, especially drug-resistant infections, continue to cause public health problems. While culture methods currently serve as the reference method for detecting and characterizing most bacterial infections, new molecular techniques provide the means for rapid, specific, and sensitive detection of pathogenic bacteria. Previously, molecular detection of bacteria focused on difficult-toculture or slow-growing bacteria; however, with the advent of more robust instrumentation, molecular assays are used to identify, detect, and track the epidemiology of drug-resistant bacteria and hospital-acquired bacteria. This review discusses select nucleic acid tests (NATs), nucleic acid amplification tests (NAATs), and their applications for detecting and characterizing bacterial pathogens. After reading this review, readers should be able to describe the theory behind various molecular methods utilized for bacterial detection and list the advantages and disadvantages of conventional culture methods versus molecular methods. Microbiology exam questions and corresponding answer form are located after the CE Update article on page 437. Accurate diagnosis of bacterial infections decreases the spread of the disease and facilitates appropriate patient management. 1-3 In addition, efficacious disease treatment reduces side effects and slows the generation of antibiotic resistance. Traditionally, culture methods facilitate the diagnosis of most bacterial infections 4,5 ; however, some bacteria are difficult to isolate, grow slowly in the culture due to stringent growth requirements, or may not grow because of prior empirical treatment of patients with antimicrobial agents. 1 In these cases, the sensitivity of culture is reduced and the time from specimen receipt to final report (ie, the turnaround time) is shortened. Hence, alternative methods capable of overcoming these limitations must be developed. Over the last 2 decades, bacterial nucleic acid sequence data (ie, genomic and ribosomal) was obtained and included in numerous databases. This information enabled the development of several new nucleic acid tests (NATs) for the diagnosis of bacterial infections. Briefly, NATs use nucleic acid primers or probes that are complementary to the nucleic acid of the target sequence of the bacteria. These primers or probes bind to the bacteria s target sequence, forming a hybrid molecule. Detection of this hybrid can be accomplished either by direct detection of the hybrid using a reporter molecule (ie, signal amplification) or enzymatic amplification of the hybrid followed by detection of the amplified product (ie, target amplification). Development of these assays, therefore, requires knowledge of the nucleic acid sequences of various isolates of the bacteria of interest. Specifically, false-positive reactions can result between genetically related species. Conversely, strains that have nucleic acid changes or variations within their target sequences can fail to hybridize with the primer or probe, resulting in a false-negative reaction. Even with these limitations, NAT assays that are more sensitive and rapid than culture have been developed (Table 1). Table 1_Comparison of Culture and Nucleic Acid Test (NAT) Methods Culture Pros High specificity Isolates can be tested for antibiotic sensitivity Enables biochemical characterization of phenotype Less expensive Does not require special workflow Does not require specialized instrumentation Permits visual inspection of colony morphology NAT Method Pros High sensitivity Rapid turnaround time Permits detection of nonviable bacteria Reduced biosafety concern (ie, bacteria is not propagated) Permits high resolution analysis of bacterial isolates for epidemiology purposes Permits detection of certain antibiotic-resistant bacteria without an initial culture Culture Cons Low sensitivity (low bacterial count) Cannot detect nonviable bacteria (ie, bacteria not viable due to sample processing, pretreatment of patient with antibiotics) Biochemical phenotype may not agree with genotype Longer time to result (turnaround time) for slow-growing or fastidious bacteria Biosafety concern (ie, bacteria is propagated) NAT Method Cons False-positive results due to cross reaction with genetically related bacteria False-negative can occur (inhibitors in sample can cause amplification to fail, genetic changes in bacterial nucleic acid prevent the primer or probe from binding) Genotype may not agree with biochemical results Requires special instrumentation or workflow Requires additional training Few tests for antibiotic sensitivity are available Downloaded 430 from

2 Image 1_Agilent chip reader. Except for the FDA-approved Mycobacterium tuberculosis molecular tests, most molecular diagnostic assays for bacteria are FDA-cleared assays. These FDA-cleared assays detect the following bacteria: Bacillus anthracis, Enterococcus faecalis, Francisella tularensis, Gardnerella sp, group A and group B Streptococci, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Chlamydia trachomatis, and Neisseria gonorrhoeae. This review focuses on some of the commercially available NATs used for the detection and characterization of common bacterial infections (ie, Chlamydia trachomatis, Neisseria gonorrhoeae, tuberculosis, nosocomial-infection-associated and antibiotic-drugresistant-associated bacteria). Chlamydia trachomatis and Neisseria gonorrhoeae Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections are the 2 most common sexually transmitted bacterial diseases in the world. In the United States alone, about 2.8 million cases of CT and 720,000 cases of NG are reported each year. 1 Nearly 79% of these infections occur in women between 15 and 24 years of age. A majority of these women (80%) do not have discernable symptoms and are the potential source for spreading the disease. 1 Therefore, routine screening for CT/NG infections is recommended in sexually active young women and men to prevent some of the consequences (such as pelvic inflammatory disease, infertility, and chronic pelvic pain) associated with chronic CT/NG infection. 6 Cell culture has been considered the reference method for the detection of CT/NG infections because of its high specificity (nearly 100%) and other advantages such as low cost and the opportunity to obtain viable organisms for antibiotic susceptibility testing and epidemiological investigation. 7 However, cell culture is not widely used for the diagnosis of CT/NG infections due to low sensitivity (70% to 80%) and frequent specimen collection and processing failures. 8,9 More than 60% of women at risk for CT/NG infections do not undergo screening due to the invasive method of collecting vaginal samples. 9,10 Noninvasive screening methods, such as urine testing and self-collected vaginal swabs, may eliminate some of the barriers to screening for CT/NG infections and have been shown to substantially increase the acceptability and convenience of screening in a variety of settings. 11,12 Patient-friendly screening methods for CT/NG might help improve adherence to current screening guidelines. 12 Nucleic acid testing may provide an additional level of sensitivity that would permit patient-friendly screening methods. Because of their enhanced sensitivity, NATs and nucleic acid amplification tests (NAATs) are the preferred assays for CT/NG diagnosis. As a result, a wide variety of NAT and NAAT assays are available for CT/NG testing. One NAT that employs probes to detect the target by forming a probe:target hybrid is the FDA-cleared Gen-Probe PACE 2 assay (Gen-Probe, San Diego, CA). This direct probe assay targets ribosomal RNA, which is present in thousands of copies per cell compared with a single-copy DNA target and thus is more sensitive. 1 Sensitivity may be further increased by using the hybridization protection assay (HPA) method for hybrid detection (Figure 1). Unlike direct-probe assays, NAAT assays enzymatically produce millions of copies of the target sequence resulting in even greater sensitivity. Because millions of copies of the target sequence are produced, special precautions must be taken to avoid contamination that could cause false-positive results. Figure 1_Detection of amplicon with DNA probes and the hybridization protection assay (HPA) technique. (A) Acridinium ester (AE)-labeled DNA probes are added and allowed to hybridize to specific target sequences within the amplicon produced in the TMA reaction. (B) Separation of hybridized from unhybridized probes is done by the addition of selection reagent, which hydrolyzes the AE on the unhybridized probes. No light is emitted in the luminometer from the unhybridized probes. (C) The AE on the hybridized probes is protected within the double helix and is not hydrolyzed by the selection reagent. Light is emitted and detected by the luminometer. Downloaded labmedicine.com from July 2008 j Volume 39 Number 7 j LABMEDICINE 431

3 Figure 2_Transcription-mediated amplification cycle (TMA). (1) Promoter-primer binds to rrna target. (2) Reverse transcriptase (RT) creates DNA copy of rrna target. (3) RNA:DNA duplex. (4) RNAse H activity of RT degrades the rrna. (5) Primer 2 binds to the DNA and RT creates a new DNA copy. (6) Double-stranded DNA template with a promoter sequence. (7) RNA polymerase (RNA Pol) initiates transcription of RNA from DNA template. (8) 100 to 1,000 copies of RNA amplicon are produced. (9) Primer 2 binds to each RNA amplicon and RT creates a DNA copy. (10) RNA:DNA duplex. (11) RNAse H activity of RT degrades the rrna. (12) Promoter-primer binds to the newly synthesized DNA. RT creates a double-stranded DNA and the autocatalytic cycle repeats resulting in a billion-fold amplification. Figure 3_Real-time PCR amplification showing high-positive, lowpositive, and negative samples and threshold for detection of fluorescence indicated. A sample with an intermediate level is seen between the high-positive and low-positive amplification curves. In addition, nonspecific amplification of non-gonorrhoeae Neisseria species has been reported with some NAAT assays. False-negative results can occur if substances are present in the sample that inhibit the enzymes. Additional amplification controls must be included in these assays to rule out this potential artifact. Several NAAT methods (ie, transcription-mediated amplification [TMA], strand-displacement amplification [SDA], and the polymerase chain reaction [PCR]) can detect CT/NG infections Transcription-mediated amplification and SDA use multiple enzymes to amplify the target molecule. Transcriptionmediated amplification is an isothermal (ie, single-temperature) amplification process that requires 3 enzymatic activities in 2 enzymes: reverse transcriptase (RT) and RNase H and RNA/ DNA polymerase (Figure 2). The RT makes a complementary DNA (cdna) copy from the RNA, and the RNase H digests the RNA. Ultimately, a double-stranded DNA molecule with a promoter sequence is generated, forming a transcriptional unit. This RNA polymerase binds to the promoter sequence, resulting in multiple single-stranded RNA copies. The RNA copies are then reverse-transcribed into DNA copies, which will serve as additional transcriptional units for the RNA polymerase. Ultimately, millions of RNA copies will be generated and detected by a luminometer. The FDA-cleared, TMA-based Gen-Probe APTIMA assay (Gen-Probe) can identify both CT and NG in a single specimen. Similar to the direct-probe PACE assay, this assay targets ribosomal RNA using primers toward the 23S rrna (CT) and the 16S rrna (NG). 13 The assay decreases the presence of enzymatic inhibitors by capturing, purifying, and concentrating the target nucleic acid in the sample preparation. Strand-displacement amplification uses 2 enzymes (ie, a polymerase and a restriction enzyme) to generate multiple copies of the target sequence. The BD ProbeTEC CT and NG assay (BD, Franklin Lakes, NJ) is an FDA-cleared assay that uses the SDA methodology. Polymerase chain reaction differs from TMA and SDA in that it uses a single enzyme and 3 different temperatures to amplify the target sequence. When the amplified product is detected after completion of the PCR cycling process, the assay is referred to as end-point PCR. The COBAS Amplicor CT/NG assay (Roche Diagnostics, Basel, Switzerland) uses this approach and is FDA-cleared. In contrast, real-time PCR is a closedtube system, which reduces the chances for contamination, and combines amplification with the fluorescent-based detection of PCR products (Figure 3). 17 Fluorescence resonance energy transfer (FRET) probes, such as Taqman or molecular beacons, can be used in real-time PCR applications. These probe-based detection methods increase the specificity of the assay but may be less sensitive if a variant sequence in the probe binding area is present. Non-sequence-specific DNA-binding d such as SYBR green are also used for the detection of amplified products. Some of the most commonly used real-time PCR instruments include the LightCycler (Roche Diagnostics, Mannheim, Germany), the icycler (Bio-Rad Laboratories, Hercules, CA), the ABI PRISM (Applied Biosystems, Foster City, CA), and the SmartCycler (Cepheid, Sunnyvale, CA). However, at the time that this manuscript was prepared, no real-time PCR CT/NG assays had been cleared or approved by the FDA. Signal-amplification methods nonenzymatically detect target:probe hybrid. An example of this method is the Digene Hybrid Capture 2 (HC2) Combo CT/NG assay (Qiagen Corporation, Germantown, MD) (FDA-cleared test) that uses antibodies specific to RNA:DNA hybrids to detect hybrid formation. 16 This technology is similar to immunohistochemical methods Downloaded 432 from

4 CE Update Table 2_Potential Problems with Signal- and Target-Amplification Methods Potential Problems Signal Amplification Target Amplification Special equipment and training required False-negative reactions due to variations in the probe/primer binding complex False-negative: Enzymes could be inhibited by substances within sample False-negative: Enzymes could have reduced activity due to poor storage False-positive reactions due to cross-hybridization with genetically related bacteria False-positive reactions due to contamination with amplified product no no no where each antibody carries an enzyme label and generates a detectable product by reacting with a substrate. Other examples of signal amplification methods include branched-chain DNA (bdna) and in situ hybridization (ISH), but no CT/NG tests are available using these methods. Table 2 summarizes the potential problems associated with target and signal amplification. Diagrams of these molecular methods can be found in the review by Sra and colleagues.17 Tuberculosis Nearly 2 billion individuals worldwide (one-third of the population) are infected with Mycobacterium tuberculosis (Mtb). In addition, 8.8 million new cases and 2 million deaths are reported each year.19 The majority of the deaths and new cases occur in developing countries. In these countries, the high prevalence of HIV infection has worsened the situation, as the risk of death is double in HIV-infected patients with Mtb than that of either disease alone. Despite this enormous global burden, case detection rates are low, posing major hurdles for controlling tuberculosis. Adding to the burden is that nearly two-thirds of tuberculosis infections occur as latent-type with many infections caused by antibioticresistant Mtb strains.20 Currently, the identification of latent Mtb infection relies on the tuberculin skin test. Likewise, the diagnosis of active disease and the detection of drug resistance continue to rely on conventional tests such as sputum-smear microscopy, mycobacterial culture, and chest radiography. As stated in the review by Cheng and colleagues, microscopy and culture may result in low detection rates when the bacterial burden is below the detection rate in sputum smears (ie, less than 104 bacilli per milliliter) or when decontamination methods reduce the number of viable bacilli.21 In addition to these factors, the time required to detect Mtb in culture ranges from 12.7 days (ie, BD Bactec 9000 MB) to 4 to 6 weeks (ie, Lowenstein-Jensen medium) depending on the culture method. Culture methods also introduce additional risks for laboratory staff by generating an abundance of viable Mtb bacilli. Two FDA-approved NAAT kits are currently available for direct respiratory specimen examination. They include the TMA-based Amplified Mycobacterium tuberculosis Direct Test (MTD; Gen-Probe) and the PCR-based Amplicor Mycobacterium tuberculosis (MTB) tests (Roche Diagnostics). These assays permit the rapid and sensitive detection of Mtb in clinical specimens. Although there is value in using molecular methods to confirm the identity of culture-positive Mtb isolates, the ability to directly detect Mtb in sputum specimens provides better clinical service and a safer work environment for laboratory professionals. Image 2_Cobas instrument from Roche Diagnostics. Epidemiology Nosocomial infections cause significant morbidity and mortality in hospital settings. In the United States alone, Downloaded labmedicine.com from Image 3_GenXpert from Gen-Probe. July 2008 j Volume 39 Number 7 j LABMEDICINE 433

5 88,000 deaths and 2 million new cases are reported annually. Risk factors for outbreaks of noscomial infections include underlying comorbid conditions such as diabetes mellitus, renal failure, and malignancies; long hospitalizations; the prior exposure to antimicrobial therapy; and the use of indwelling catheters. The most common nosocomial infections are caused by vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus (MRSA), and extended-spectrum beta lactamase-producing strains of Escherichia coli. 22,23 An integral part of any rational prevention and control strategy requires establishing the epidemiology of nosocomial infections. Previously, epidemiological analysis of nosocomial infections relied on phenotypic characteristics that lack the high discerning resolution of molecular methods. The reference method for discerning relatedness between strains is pulsed-field gel electrophoresis (PFGE) because of its high degree of isolate differentiation and reproducibility. 24,25 In this method, bacterial genomic DNA is enzymatically cleaved into different-sized fragments. By changing the direction of the electric field, large DNA fragments that would normally appear as a smear will take on a bar-code appearance. PFGE has been applied to at least 38 different pathogen groups; however, this technology does have some limitations that include (1) long turnaround time, (2) unsatisfying comparability between laboratories, and (3) banding patterns that are only partially informative with regard to phylogenetic relationships. Ribotyping is a similar approach that uses restriction enzymes with standard gel electrophoresis system and Southern analysis. This process has been partially automated using the RiboPrinter System (Dupont Qualicon, Wilmington, DE). An alternative to this method is multiple-locus variable-number tandem repeat analysis (MLVA) that uses PCR to amplify repeated regions within the bacterial genome. This method enzymatically produces multiple PCR products of various lengths. The resulting PCR products form a pattern, and this data can be integrated with an Internet-based database to assign MLVA types Another example of this approach is the Bacterial Barcode assay that integrates PCR product sizes with its Diversilab system software for data analysis. This approach enables rapid identification of resistant type as well as the source of origin (Figure 4). Sequencing methods characterize the genetic makeup of an organism with the highest level of resolution and, therefore, have the greatest ability to differentiate between organisms. 28 Briefly, sequencing identifies the exact order of the 4 nucleotide bases (ie, datp, dttp, dctp, and dgtp) using a combination of fluorescently labeled chain-terminating nucleotides and high-resolution fragment analysis. Multilocus sequence typing (MLST) is a new molecular technique that was developed initially for Staphylococcus aureus but has been applied to Neisseria meningitidis, major hypervirulent clones of Streptococcus pneumoniae, and Enterococcus faecium. 29,30 This technique characterizes bacterial isolates on the basis of allelic profiles generated by determining 450 base-pair internal sequence fragments of 7 housekeeping genes. Multilocus sequence typing has been successfully used for the study of molecular epidemiology and evolution of virulence of various bacterial species. Despite the high accuracy and portability of MLST, the cost and labor required for MLST are significant. Other sequencing-based methodologies include the MicroSeq 16S integrated system (Applied Biosystems, Foster City, CA). This sequence-based approach targets conserved regions in the 16S encoding gene and uses software and a database to assist in the identification of bacteria. Other databases useful for sequence comparison include GenBank, RDP-II, and RIDOM. These assays would primarily be used to identify bacteria where the biochemical profile is inconclusive. Whether a laboratory uses PFGE or sequencing-based assays to determine the source of infection, data derived from these molecular assays is Figure 4_Demo report with graph overlay: Samples have color-coded boxes by location. Up to 10 demographic fields can be associated with each sample. The similarity matrix was derived using the Pearson correlation coefficient. The dendrogram was then created using UPGMA. The computer-generated gel-like images provide the fingerprint. The software has an interactive report that allows users to view graph overlays of samples by clicking on boxes of the similarity matrix. Downloaded 434 from

6 useful for establishing patient isolation procedures, monitoring outbreaks, and reducing cost. Antibiotic Resistance Testing Susceptibility testing by agar plate diffusion or broth microdilution remains the reference method for the detection of drug resistance. These approaches require an initial culture followed by a secondary culture of the isolate. Because these multiple culture steps require additional incubation time, real-time PCR and microarrays are being developed and implemented. Of particular importance is methicillin-resistant Staphylococcus aureus, which has become a public health risk. A recent report of the MRSA prevalence in United States health care facilities cited an average frequency of 46.3 MRSA infections/colonizations per 1,000 patients. Five states (Delaware, Maine, New Hampshire, New York, and South Carolina) had prevalence rates greater than 60 per 1,000 patients. 31 Some of the FDA-cleared molecular assays available for testing antibiotic resistance include the real-time PCR-assay-based BD GeneOhm StaphSR Test (GeneOhm Sciences, San Diego, CA), Cepheid Xpert MRSA test (Cepheid, Sunnyvale, CA), and IDI-MRSA test (Infectio Diagnostic, Quebec, Canada). Efforts are currently underway by some hospitals to reduce their prevalence of hospital-acquired MRSA by screening patients using these rapid tests. Glossary of Initialisms and Terms bdna: Branched-chain DNA. A signal-amplification molecular diagnostic method. cdna: Complementary DNA synthesized from RNA by reverse transcriptase. CT: Chlamydia trachomatis. Bacteria that causes the sexually transmitted disease chlamydia. CT/NG: Chlamydia trachomatis/neisseria gonorrhoeae. datp: 29-deoxyadenosine 59-triphosphate. One of 4 nucleotide bases present in DNA. dctp: 29-deoxycytidine 59-triphosphate. One of 4 nucleotide bases present in DNA. dgtp: 29-deoxyguanosine 59-triphosphate. One of 4 nucleotide bases present in DNA. DNA: Deoxyribonucleic acid where deoxyribose is the sugar moiety. dttp: 29-deoxythymidine 59-triphosphate. One of 4 nucleotide bases present in DNA. FDA: Food and Drug Administration is an agency of the U.S. Department of Health and Human Services responsible for regulating food, vaccines, cosmetics, veterinary medicine, drugs, blood products, biological medical devices, and medical devices. FDA-approved: A medical device or assay that has been certified through the premarket approval (PMA) process. This process is for the most stringently regulated category of medical devices (ie, class III devices) and usually pertains to devices where insufficient information exists to ensure safety and effectiveness. FDA-cleared: A medical device or assay that has been certified by the Food and Drug Administration through the premarket notification or 510(k) process. This process requires that the device have substantial equivalence to another legally U.S. marketed device. FRET: Fluorescence resonance energy transfer. A process that generates a fluorescent signal. HIV: Human immunodeficiency virus HPA: Hybridization protection assay. An assay that enhances the sensitivity of detecting hybrid molecules. ISH: In situ hybridization. A signal amplification method for detecting nucleic acids in the context of tissue morphology. MDR-TB: Mycobacterium tuberculosis that is drug-resistant to 2 or more main first-line drugs (ie, isoniazid and rifampicin). MLST: Multilocus sequence typing. A process for characterizing bacterial isolates by determining the sequence of portions of multiple housekeeping genes. MLVA: Multiple-locus variable-number tandem repeat analysis. A process for characterizing bacterial isolates by generating multiple PCR products of different lengths. MRSA: Methicillin-resistant Staphylococcus aureus. Mtb: Mycobacterium tuberculosis. The bacilli that causes tuberculosis. NG: Neisseria gonorrhoeae. The bacteria that causes the sexually transmitted disease gonorrhea. NAAT: Nucleic acid amplification test. NAT: Nucleic acid test. PCR: Polymerase chain reaction. Target amplification molecular diagnostic method using a thermostable enzyme and an instrument that can cycle between numerous heating and cooling steps. PFGE: Pulsed-field gel electrophoresis. A method used to separate large DNA fragments by alternating the direction of the electric field. RNA: Ribonucleic acid where ribose is the sugar moiety. RT: Reverse transcriptase enzyme. An enzyme that synthesizes complementary DNA from RNA. RNase H: Nuclease that specifically degrades only the RNA strand in RNA-DNA hybrids. SDA: Strand-displacement amplification. Target amplification molecular diagnostic method that generates large numbers of DNA TMA: molecules using 2 enzymes and 1 temperature. Transcription-mediated amplification. Target amplification molecular diagnostic method that produces large numbers of RNA molecules using multiple enzymes and 1 temperature. XDR-TB: Extensively drug-resistant Mycobacterium tuberculosis (also referred to as extreme drug resistance); MDR-TB that is also resistant to 3 or more of the 6 classes of second-line drugs. 16S rrna: Ribosomal ribonucleic acid that has a sedimentation rate equal to 16 Svedberg units and comprises the majority of the 30S ribosomal subunit for prokaryotes. 23S rrna: Ribosomal ribonucleic acid that has a sedimentation rate equal to 23 Svedberg units and constitutes the majority of the 50S ribosomal subunit for prokaryotes. Downloaded labmedicine.com from July 2008 j Volume 39 Number 7 j LABMEDICINE 435

7 Another drug-resistant bacteria posing a public health risk is multiple-drug-resistant (MDR) and extensively-drug-resistant (XDR) Mtb. While not FDA approved, systems for detecting mutations associated with rifampin and isonazid resistance are available now from several companies and include a microchip approach (Autogenomics, Carlsbad, CA), a line probe assay (Innogenetics, Alpharetta, GA), and the GenoType MTBDR assay (Hain Lifesciences, Nehren, Germany). These array-based methods use PCR followed by target-specific probes to detect drug-resistant Mtb isolates on a solid matrix. The availability of only a few FDA-cleared molecular assays toward the same drug-resistant bacteria illustrates that molecular-based detection of drug-resistant bacteria is in its infancy. However, molecularbased nucleic acid tests that can rapidly identify drug-resistant isolates promise to improve treatment efficacy and manage the spread of these dangerous drug-resistant bacteria. Conclusion When determining the best molecular or nonmolecular diagnostic application for a particular epidemiologic or patient care question, the strengths and limitations must be considered by the laboratory. These include identifying the cost and benefit of training personnel, acquiring the specialized equipment, understanding possible causes for false-negative or -positive results, and gaining the necessary skills to interpret complex results from epidemiology assays. For these reasons, molecular assays for diagnosing bacterial infections are mainly confined to clinical microbiology and molecular diagnostic laboratories. Widespread use of molecular methods for routine diagnosis of bacterial infections will require advances in instrument automation in order to be cost-effective for the typical laboratory. LM Acknowledgments: The authors acknowledge Gen-Probe and Bacterial Barcodes for providing images and Jeanelle Vaughn for generating real-time PCR figures. 1. 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