principles of molecular microbiology testing methods - Infectious ...

THE ROLE OF THE CLINICAL MICROBIOLOGY LABORATORY

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PRINCIPLES OF MOLECULAR MICROBIOLOGY TESTING METHODS Donna Wolk, PhD, MHA, MT; Shawn Mitchell, MMSc, MT; and Robin Patel, MD, FRCP(C)

Recent advances in molecular testing methods are changing the practice of clinical microbiology and infectious disease. In the past decade, nucleic acid technologies have complemented conventional culture, antigen-based, and antibody-based methods for the detection, identification, and epidemiological analysis of infectious microorganisms.", 76, Io4, lo6,lo7,'09, 118 Until recently, molecular approaches to infectious disease diagnosis have been limited to the detection of slow-growing, difficultto-cultivate, or uncultivatable microorganisms;29~ 83, 85 however, advances in testing formats, including automated nucleic acid extraction instrumentation and rapid polymerase chain reaction (PCR)/target detection formats, now make molecular diagnostic testing adaptable for use in many clinical laboratories and applicable for a variety of common pathogens. Some assays offer the capability of detection, quantification, and mutation screening in the same testing format, and may be useful in screening for antimicrobial resistance-associated mutations.18,73 The number of commercially available assays continues to expand, and scores of "home-brewed" molecular assays continue to develop. Examples of infections for which the clinical use of molecular diagnostics is relevant include group A streptococcal pharyngitis,", 36, 79,lo* herpes simplex virus (HSV) encephaliti~,4~< 58, lo5central nervous system (CNS) toxoplasmosis, progressive multifocal leukoencephalopathy (PML), hepatitis C virus (HCV) infection? 22 pulmonary tuberculosis (TB)," 13, 16, 25, 86,

From the Division of Clinical Microbiology (DW, PSM, RP) and the Division of Infectious Diseases (RP), Mayo Clinic, Rochester, Minnesota

INFECTIOUS DISEASE CLINICS OF NORTH AMERICA VOLUME 15 * NUMBER 4 * DECEMBER 2001

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and urogenital infections caused by Chlamydia trachomatis and Neisseria gonorrkoeae.lo,33, 44,89 Molecular tools are also used for quantification of pathogens, such as HIV-1, to monitor therapy, or disease outcome. In addition, studies of the genomic differences and similarities among microorganisms of the same type are used to determine the epidemiology of disease in public health outbreaks and in nosocomial infect i o n ~ .lo8 ~~, Nucleic acid amplification technologies like PCR are important in the diagnosis of infectious disease agents, because, in contrast to human genetic disease (e.g., chromosomal polymorphism) detection where the amount of DNA is plentiful, the amount of microbial nucleic acid found in human infections may be limited. PCR was one of the first nucleic acid amplification techniques to be broadly applied to the molecular detection of microorganisms, and it is becoming standard methodology in clinical and research microbiology laboratories. Since the original description of PCR6* and the first report of Tkermus aquaticus (Taq) DNA polymeraseg1(the enzyme used for PCR), numerous technological advances have facilitated the application of this methodology to routine clinical molecular microbiology diagnostics. In addition, a number of non-PCR amplification molecular diagnostic methodologies are also used. Molecular methods continue to be valuable research tools to identify new infectious causes of disease and to establish infectious links to acute or chronic disease states. Adaptations of research methods will continue to impact testing in clinical laboratories. For example, in the future, gene expression profile analysis (i.e., mRNA assays) of humans infected with specific pathogens may be used to help determine the nature of the infection, or predict response to these pathogens. While the potential applications of molecular research to clinical microbiology are innumerable, it is important to understand that molecular tests performed in a research setting differ in both style and substance from their diagnostic laboratory counterparts. As technology advances, additional commercial, Food and Drug Administration (FDA)-approved methods will become available and eventually enable even the smallest laboratory to apply molecular technologies to the detection of microorganisms. In the meantime, efforts should continue to increase understanding of the strengths and limitations of these new methods. Molecular diagnostic tests may enhance diagnostic capabilities, but they should be interpreted within clinical context and on the basis of individual laboratory performance. Extensive clinical research and strict adherence to guidelines for method validation are necessary to compare new molecular diagnostic techniques with existing methodologies, to validate new technology when comparable conventional techniques are unavailable, and to determine a method's clinical ~ti1ity.l~ Results derived from these studies are vital to the practical implementation of molecular tests in the clinical laboratory and to the assessment of their clinical utility and cost-benefit for patient testing.52 Although molecular diagnostic tests have traditionally been more expensive than conventional diagnostic techniques, the ease with which some 95, loo, 117

PRINCIPLES OF MOLECULAR MICROBIOLOGY TESTING METHODS

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molecular tests can now be performed and the rapid results generated by these methods can provide timely diagnosis and translate into overall savings. For example, a rapid automated molecular test method may replace labor-intensive cell culture methods used to detect viruses. Cost savings may be realized, because rapid diagnosis may prevent invasive diagnostic procedures (e.g., brain biopsies in patients with HSV encephalitis), limit unnecessary or potentially toxic empiric antimicrobial therapy, and shorten hospital stays in expensive isolation rooms (e.g., for patients with suspected pulmonary TB). Earlier detection of microbes may also limit the spread of nosocomial and community-acquired infections. PROBE HYBRIDIZATION METHODS Nucleic acid probes are segments of DNA or RNA that can be labeled with enzymes, antigenic substrates, chemiluminescent moieties, or radioisotopes, and can bind with high specificity to complementary sequences of nucleic acid. For many microorganisms, probes alone do not have optimal sensitivity for direct detection of microorganisms in clinical specimens. Nucleic acid amplification technology can overcome this lack of sensitivity. Oligonucleotide probes (usually defined as probes with fewer than 50 bases) are integral parts of several of the in vitro amplification techniques to be discussed later. Oligonucleotide probes can, under stringent reaction conditions, detect the change of a single nucleotide within a given nucleic acid sequence. These probes can also be used (in some instances) for direct detection of microorganisms in clinical specimens or for identification of microbes isolated by culture. Probebased commercial kits, such as the PACE2 from Gen-Probe (San Diego), directly detect C. trachornatis and N.gonorrhoem bacteria present in clinical samples, producing results that are generally equivalent to culture techniques in much less time. Use of such kits is described e1sewhere.l' In situ hybridization, common in pathology laboratories, is a technique that uses intact cells (e.g., in formalin-fixed, paraffin-embedded tissue) containing specific DNA or RNA as targets for hybridization with labeled nucleic acid probes. The technique allows for visualization of infected cells within the structure of the tissue itself and allows for association of hybridization results with other morphologic changes in the tissue. The sensitivity of this method may be limited. QUALITY ISSUES

Molecular techniques developed as research tools have been applied as "home-brewed" or "in-house" developed assays in clinical microbiology laboratories. No universal standards exist for these assays. In 1995, the National Committee for Clinical Laboratory Standards (NCCLS) published guidelines for molecular diagnostic methods" and empha-

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sized that the guidelines did not contain standards, because molecular methods were quickly evolving. In 2001, guidelines, as opposed to standards, still appear to be more appropriate. Strategies to improve the quality and partial standardization of molecular testing include labora20* 66 proficiency surveys forwarded tory requirements for certifi~ation,'~, and graded by an objective third party, and FDA-approval of commercial assays. Currently FDA-approved molecular microbiology assays are listed in Table 1. As with any laboratory assay, not all FDA-approved molecular assays have identical performance characteristics. Although a review of the clinical performance of each assay is beyond the scope of this article, certain universal parameters are applicable, and their definitions are important to understanding and interpreting molecular test results. For molecular microbiology testing, validation data should be available to detail both the analytical and clinical specificity and sensitivity for every "home-brewed" and commercial assay. For molecular tests, analytical specificity refers to the ability of the assay to detect only the organism or analyte it purports to measure. Clinical specificity is the proportion of specimens from patients who do not have a specified clinical disorder and whose test results are negative. Analytical sensitivity, also called lower limit of detection, refers to the lowest number of organisms that can be reproducibly detected by the assay. Clinical sensitivity is the proportion of specimens from patients who have a specified clinical disorder and whose test results are positive. Importantly, a molecular diagnostic assay with a high analytical sensitivity may not provide adequate clinical sensitivity if false-negative results occur, because the target nucleic acid copy number in the clinical specimen is low. For quantitative molecular microbiologic diagnostic testing, several other terms are relevant. Linear range refers to the quantitative span over which the assay provides results detecting a direct relationship between the input target concentration and the output signal. The upper and lower limits of quantification reflect the upper and lower ends of the linear range. Importantly, the lower limit of quantification of a quantitative assay may be higher than the analytical sensitivity of a qualitative assay. Result interpretation can be confusing. For example, the lower limit of quantification of the ~ ~ u n t i t u t i vHCV e RNA assay, the HCV RNA 3.0 bDNA assay (Bayer Corporation, Tarrytown, N.Y.), is 520 IU/mL, whereas the lower limit of detection of the qualitative HCV RNA assay, the COBAS AMPLICOR'" HCV Test, version 2.0 (Roche Diagnostics Corporation, Indianapolis, Ind.), is 50 IU/mL. In an effort to standardize quantitative testing, laboratory collaborations with the World Health Organization (WHO) have established the WHO International Standards, standard reference materials with concentration expressed as IU/mL, which can be used to calibrate, validate, and compare quantitative molecular assays. Three quantitative standards Additional informaexist: HCV, hepatitis B virus (HBV), and HIV-1.65,92-94 tion can be found by contacting the National Institute for Biological Standards and Controls (NIBSC) at hftp://www.nibsc.ac.uk.

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Abbott Laboratories http://www.abbottdiagnostics.com

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*Culture confirmation assays not listed tFood and Drug Administration approved as of July, 2001

Bayer http://www.bayerdiag.com/index3.html

bDNA

I1 Hybrid CapturemTM

BDProbe Tecm

TMAm

AmplifiedTM

bioMeneux (formerly Organon Teknika) http://www.nuclisens.corn Gen-Probe http://www.gen-probe.com Bayer http://ww.bayerdiag.com/products/nad/ qualitivehcvrna.htm1 BD Biosciences http://www.bd.com/biosciences Digene http://www.digene.com

Nuclisensm

Manufacturer

Roche Diagnostic Systems http://www.roche.com/diagnostics

AMPLICORm and AMPLICOR MONITORm

Test

C. trachomatis" N . gonorrhoeae* Human papilloma virus* CMV (peripheral white blood cells)* HBV HN-1 HIV-1 HBV HCV C. tmchomatis* N. gonorrhoeae*

M . tuberculosis* C. trachomatis* HCV

Mycobacterium tuberculosis* Chlamydia trachomatis* Neisseria gonorrhoeae* HN-1' Human T cell lymphotrophic virus types I and I1 Hepatitis B virus (HBV) Hepatitis C virus (HCV)" Cytomegalovirus (plasma) (CMV) HIV-1 CMV 67 mRNA (whole blood)*

Application

Qualitative ligase chain reaction (LCR)

Quantitative branched DNA @DNA) assay

Qualitative strand displacement amplification (SDA) Qualitative or quantitative (varies with target) hybrid capture assay

Qualitative or quantitative (varies with target) nucleic acid sequence-based analysis (NASBA) Qualitative transcription mediated amplification (TMA) Qualitative TMA

Qualitative or quantitative (varies with target) polymerase chain reaction (PCR) Can be automated on the COBAS instrument

Detection

Table 1. EXAMPLES OF COMMERCIAL AMPLIFICATION ASSAYS FOR MOLECULAR DIAGNOSIS OF MICROBES INFECTING HUMANS (UNITED STATES, 2001)t

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Other important components of quantitative molecular assays include precision, accuracy, and the tolerance limit. Precision refers to the agreement between replicate measurements of the same material. Accuracy refers to the ability of a method to reliably determine the true value level of the particular target. The tolerance limit is the difference between two sequential samples that can be significantly different and is the sum of the biological variation in quantitation and the intra-assay variation of the assay. For example, for HIV-1 quantitative assays, when biological and intra-assay variation are considered, increases or decreases in HIV concentration of at least three-fold typically reflect biologically relevant changes in the level of viral replication. Of no less importance is the sensitivity of the assay to detect the target in spite of nucleic acid sequence variation of the target, which may occur in different strains of the organism. For example, HIV-1 RNA viral load may not be equally quantified among all subtypes using certain assays.12, lo3 A thorough understanding of molecular microbiology assay parameters is important for interpretation of laboratory results and for comparison of results generated with different assays.23 For each molecular diagnostic assay, criteria for the appropriate specimen must be defined. These include the optimal specimen source, specimen volume, collection method, transport and storage conditions, and specimen longevity. The sensitivity and specificity of an assay may vary considerably if any of these conditions are altered. Typically, molecular microbiologic assays are clinically validated using one or more specimen source(s) (eg., blood or cerebrospinal fluid [CSF]) that may contain the target organism. The choice of specimen plays a key role in the performance and interpretation of test results. Even after deciding that the desired specimen for a given target is blood, there remains a number of specimen fraction options including plasma, serum, whole blood, and various leukocyte fractions. As quantitative molecular assays are becoming more common, careful definition of the optimal specimen is gaining importance. For example, the clinical significance of viral loads will vary depending, among other factors, on the specific specimen assayed. Plasma or serum is preferred over whole blood or leukocytes when the target microorganism is predominantly extracellular (e.g., HBV DNA, HCV RNA, and HIV-1 RNA). By contrast, the ideal blood fraction for molecular assays for quantitation of cytomegalovirus (CMV) in the blood of transplant recipients has not yet been determined, although many different fractions have been used. Adding complexity to this issue, the concentration of target DNA in various blood fractions may also vary depending on the method of storage and extraction. The volume of specimen from which nucleic acid is extracted also varies considerably. Most molecular diagnostic tests use specimen volumes of 200 p.L or less; however, in some cases larger volumes may be required. This issue is critical when the number of infectious organisms per test volume is small. All things considered, lack of standardization in specimen processing is an important reason why results may be difficult to compare from laboratory to laboratory. Just as antibiotic treatment may render a conventional blood culture


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falsely negative, inhibitory substances (e.g., bilirubin, hemoglobin, lipids, heparin), may interfere with various steps in molecular diagnostic assays. Inhibitors can be present in patient specimens or introduced during specimen collection or processing. Their interaction with nucleic acid or critical enzymes, especially DNA polymerases, can prevent amplification of the target. Likewise, inhibitors may remove reaction components (e.g., metals), which affect enzymatic substrates, resulting in false-negative results. The presence of such inhibitors can be determined by several methods. One approach is to incorporate amplification controls into the PCR assay design. Spiking the patient’s specimen with known target DNA or RNA and assaying t h s ”spiked” specimen along with the native patient specimen ensures that a negative result is truly negative and not the result of assay inhibition. When spiked specimens are assayed, the negative predictive value of the assay is enhanced. Another approach involves the use of internal amplification controls in a single sample. These controls amplify concurrent to the target nucleic acid and are used to detect the presence of inhibitors and can be used to evaluate the quality and quantity of nucleic acid. To assess the adequacy of specimen collection, human housekeeping genes like human p-globin or interleukin-2 can be included to assess the presence of human cellular DNA. As an example, if a throat specimen contains human DNA, this may assure that an adequate specimen was collected. Specimen preparation techniques are an important facet of the overall usefulness of molecular technologies used in clinical microbiology laboratories. Optimal specimen preparation will efficiently release the nucleic acid from the microbe, while preserving the integrity of the nucleic acid target, removing inhibitors, sterilizing the specimen of viable organisms, concentrating the target nucleic acid into a small volume (if appropriate), and placing the target into an aqueous environment suitable for amplification. Many specimen preparation methods exist, but may require considerable technical skill, because they are labor-intensive, non-standardized, and prone to manipulation steps where template cross-contamination may occur. Specimens are commonly treated with chaotropic agents, alkaline pH, freeze-thaw cycles, sonication, heat, detergents, and/or proteolytic enzymes to disrupt the cell membranes and release nucleic acid.’O* Nucleic acid is commonly extracted with organic solvents, and/or adsorption to silica in the presence of chaotropic agents such as guanidinium salts, and/or precipitated in the presence of salts and cold isopropanol. Novel nucleic acid capture technologies include automated silica-based membrane capture technologies and magnetic bead capture of nucleic acids. Automation of specimen preparation is expected to provide rapid, cost-effective, and consistent results. Results for automated nucleic acid extraction are promising. Like some manual nucleic acid extraction methods, however, PCR inhibitors may coextract with nucleic acid and reduce the clinical and analytical sensitivities of the molecular assays.*” 43, Several new automated extraction instruments are available (see Table 4).The ability to interface with high throughput PCR systems is an

‘FRET = fluorescent resonance energy transfer tCCD = charge coupled device $NET = National Institute of Standards and Technology SLED = Light emitting diode

Smartcycler@System

Corbett Research http://www/corbet tresearch.com Phenix Research Products h t tp://www.phenixl.cam Cepheid ht tp://www.smartcycler.com http:/wwzu.cepheid.com. Fisher Scientific http://www2$shersci. comlmain.j s p

32-72/ Run

1&96 f Run Ceramic heating plate

Proprietary plastic reaction tubes

96/Run

Thermal cycler

Microplate, eight strip tubes or PCR tubes

Stratagene

MX4000TM Multiplex Quantitative PCR System Rotor Gene Rapid cycling with ambient air cooling

32/Run

Rapid cycling with ambient air cooling

Glass reaction tubes

Roche Molecular Biochemicals http://www.roche.com/diagnostics

Lightcyclerm

Thin-walled plastic PCR tubes or strip tubes

9&384/ Run

Thermal cycler

Plastic microplate or PCR tubes

Biorad http://www.bio-rad.comlicycler

iCycler iQ System

http://www.stratagene.com/qgcu

96 Run

Reaction Capacity

Thermal cycler

Cycling

Dehydrated reaction components in plastic microplate

Format

Applied Biosystems ht tp://www.appliedbiosystems.com

Source

GeneAmpa5700 and PRISM@ 7700 Sequence Detection Systems

Instrument

LED light source with silicon photodetectors 16 independent &COREm modules per sample block Up to four targets per reaction

Designed for fixed hybridization temperature Potential for at least three to four probe sets per reaction Fiber optic detection by way of CCD Endpoint analysis of real-time data Passive internal reference and internal PCR controls Hybridization temperature can vary Light intensifier technology and CCDt Real-time monitoring capabilities NISTS -traceable temperatures Gradient heating option Dual sample blocks for independent runs Hybridization temperature can vary Potential for one to two probe sets per reaction Light detected by luminometer Real-time monitoring capabilities Potential for up to four probes per reaction Internal control kits available Broad emission detection range Molecular beacons only Two independent LED§ light sources

Comments

Table 2. EXAMPLES OF AUTOMATED CLOSED-SYSTEM QUANTITATIVE REAL-TIME PCWHYBRIDIZATION INSTRUMENTS THAT USE FRET*, TAQMANTM,AND MOLECULAR BEACON PROBES


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important advance of many new automated extraction systems. For example, the Roche MagNA Pure (Roche Molecular Biochemicals, Indianapolis) and the ABI PRISM 6700 instrument (Applied Biosystems, Foster City, Calif.) integrate with the Lightcycler PCR instrument (Roche Molecular Biochemicals, Indianapolis) and the ABI PRISM '' 5700 and 7700 (Applied Biosystems, Foster City, Calif.), respectively, and can be used to automate or partially automate both extraction and PCR setups (Table 2). TM

TM

TARGET NUCLEIC ACID AMPLIFICATION METHODS Polymerase Chain Reaction

PCR is the most widely used nucleic acid target amplification technology. For PCR, a DNA sequence (template) is amplified in a buffered reaction solution containing oligonucleotide primers, thermostable DNA polymerase, deoxynucleotides triphosphates (dNTP), and magnesium or manganese ions. Other additives, used to enhance amplification, are common but not essential for the reaction to occur. The reaction solution is placed into a thermal cycler, which heats and cools the reaction components, exposing them to consecutive cycles of varying temperatures. In each temperature cycle, three steps occur: denaturation (heating to high temperatures to separate DNA into single strands), primer annealing (lowering the temperature to allow for primers, synthetic oligonucleotide strands designed with a sequence that is complementary to the ends of the original target gene sequence, to anneal to the single stranded DNA and create a partial double strand), and primer extension (addition of dNTPs to the ends of the bound primers by DNA polymerase, thereby creating a new synthetic piece of double-stranded DNA [the amplimer or amplicon], which is complementary to the original template strand). The annealing and extension steps can be combined into a single step for some reactions. With the exception of the first cycle, the number of amplicons will theoretically double with each cycle (Fig. l),resulting in an exponential increase in the quantity of amplicon, until the reaction reaches a plateau phase caused by depletion of reaction components. After a 36-cycle amplification, amplicon is present in the reaction tube at a theoretical concentration of approximately 6.9 X 1O1O copies per each original template (Fig. 1). The exact concentration of amplicon is lower when the reaction efficiency is less than 100%. Traditionally, amplicons have been detected and identified by a variety of methods. Many of these methods are manual, require subsequent manipulation of the PCR amplicon, and add substantial risk of contamination to subsequent PCR reactions. One of the first methods developed, gel electrophoresis, uses electrical current in an agarose or polyacrylamide matrix to separate DNA fragments by size. Visualization of DNA fragments is possible by staining the gel fragments with DNA binding dyes (e.g., ethidium bromide). Using this methodology, ampli-

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