Molecular Diagnostics in Large Animals

Christian M. Leutenegger • Nicola Pusterla

Of the four classes of organic molecules that compose the basic physical structure of all living beings, nucleic acids are the only molecules that carry replicable instructive information.

The amplified era of molecular diagnostics for infectious diseases began in 1983 when Dr. Kary Mullis of Cetus Corpora­tion first conceptualized the polymerase chain reaction (PCR). He went on to win the Nobel Prize in Chemistry for this revolutionizing technology 10 years later.^1,2 Since the inception of PCR technology, infectious disease diagnostics have been at the forefront of molecular medicine, with the promise of detecting contagious pathogens in a safer and more sensitive way to aid in controlling their spread. Infectious disease testing is expected to continue to dominate the molecular diagnostic market for large animals in the foreseeable future. The use of amplified and nonamplified tests to assay the molecular makeup of the host rather than the pathogen is growing. Whereas cancer diagnostics is in its infancy for large animal testing, parentage and forensic testing together with single-nucleotide polymorphism (SNP) detection using in situ hybridization (ISH), fluorescent in situ hybridization (FISH), and sequencing technologies is gaining importance in assessing the presence and prognosis of genetic diseases.

The ability of molecular diagnostic assays to sensitively and specifically detect the primary cause of a disease, with short turnaround time, is giving significant advantages to disease diagnostics.

Rapid results have tremendous clinical value in curtailing infection. Molecular methods can provide results in hours compared with days for culture-based or alternative technologies. The high sensitivity and specificity of molecular testing are

particularly valuable in early determination of infection and make it advantageous compared with antibody and antigen testing without amplification techniques. In most cases pathogens enter the host and replicate exponentially; early detection provided by the higher sensitivity allows for earlier clinical intervention and a better prognostic outlook.

• Superior sensitivity and specificity compared with most immunoassays

• Automated platforms that significantly increase throughput

• Quantitative assessment of viral load, which is clinically useful

• Fast turnaround time that speeds detection and reduces overall costs

• Simultaneous analysis of multiple analytes

• Standardized environment with the potential for full com­parability across laboratory systems

• Ability to tightly quality control and monitor all aspects of the test workflow, including sample quality, DNA and RNA quantity, contamination situation during extraction and in the laboratory in general, presence of inhibition, and per­formance of the PCR test and reagents

Technologic Superiority of Molecular Tests

The relative technologic superiority of molecular methods makes them likely to partially replace other types of more conventional methods, such as culture-based tests or certain direct antigen and antibody tests to determine the presence of an infectious pathogen in a sick animal. Whereas antibody testing is more of a broad screening tool, molecular diagnostics allow accurate detection of the genome of a pathogen in a clinically sick animal. Culture-based tests have a turnaround time of days and are labor intensive. In addition, certain infec­tious agents are difficult to culture or are biohazardous for laboratory personnel (e.g., Mycobacteria species, fungi, Myco­plasma species, Lawsonia intracellularis). In such instances, molecular detection of selected pathogens has already supplanted conventional culture.

Rapid and High-Throughput Applications Promote Molecular Tests

Rapid and specific detection of infectious disease agents is crucial for the prevention or containment of outbreaks.

Increasing Adoption of Molecular Tests by University and Commercial Laboratories

Since the advent of molecular platforms in the diagnostic laboratory, most molecular tests in veterinary medicine are performed by several commercial and public laboratories. Unlike the market in human molecular diagnostics, which is dominated by four large players (Roche, Bayer, Gen-Probe, and Abbott), veterinary molecular diagnostics show significant fragmentation in particular with many university diagnostic laboratories. However, the transition to the more advanced real-time PCR platform within the past 5 to 7 years and the abandonment of conventional PCR tests by many laboratories has led somewhat to a concentration process in the veterinary diagnostic landscape for a good reason: real-time PCR is the more advanced method and is considered a fully matured technologic platform with great incentives for automation, operational efficiency, and affordability.

Simultaneous Testing of Multiple Pathogens

Parallel testing of multiple infectious agents in highly stan­dardized platforms is a central advantage of molecular assays; it essentially allows several tests for both DNA and RNA pathogen targets to be performed simultaneously on a single sample, therefore condensing the medical message obtained on a given diagnostic sample. This development is a noteworthy driver for molecular diagnostics because it allows acquisition of more meaningful data from a single sample. This so-called panel strategy allows an efficient workup of complex clinical syndromes with general symptomatology that do not allow an etiologic diagnosis based on clinical signs.

Indications for Use of Polymerase Chain Reaction Assays for Infectious Diseases

Diagnostic tests such as PCR assays offer the potential for fast and accurate determination of the presence of an infectious agent, which can lead to an improved clinical outcome because of the faster initiation of more etiologic-based treatment and the possibility for quantitative treatment monitoring.

Key indications for use of a molecular test are the speed and accuracy of molecular assays. In comparison with traditional culture for bacteria, rickettsial organisms, fungi, and viruses, molecular testing offers direct detection of the target pathogen within a fraction of the time. Owing to its speed, PCR assays have the potential to replace traditional culture methods for infectious agents that are difficult to culture or are not cultivable at all.

Compared with serology testing, molecular tests offer the advantage of detecting an infectious agent before an immune response occurs and a detectable antibody titer is developed. Immunoglobulin M (IgM) analysis in certain applications (such as WNV) can alleviate this problem; however, molecular testing methods are more reliable in picking up early virus replication and allow a faster epidemiologic assessment and earlier patient treatment.

In certain instances, for the discrimination of a clinically relevant EHV-4 infection from a latent infection, the detection of viral DNA is not informative. In such cases the quantitative assessment of viral loads is necessary.³ Similarly, the choice of RNA or DNA affects the ability to distinguish disease (active virus replication and production of viral transcribed RNA) from nondisease (viral DNA of latently infected cells) status.

Molecular Biology Technologies

The main methodologies used for molecular diagnostics include the following:

• Nucleic acid capture, probe hybridization

• ISH, FISH

• Isothermal amplification of nucleic acids⁴

• Transcription-based amplification methods

• Signal amplification by branched-chain DNA

• Qualitative pathogen identification using nucleic acid amplification

• Quantitative assays

• Genotyping assays

• Genotype sequencing assays

• Multianalyte testing panels

Genotyping assays are typically used to test for known mutations associated with inherited genetic conditions such as hyperkalemic periodic paralysis (HYPP) in horses, an autosomal dominant condition that causes potassium-induced attacks of skeletal muscle paralysis. Many of these assays depend on sequence-specific probes designed to hybridize with known genetic variations. For infectious agents, genotyping or specia­tion is achieved by using highly specific PCR- or hybridization­based assays or by restriction enzyme fragment length polymorphism (RFLP) assays. In these assays, amplified material is digested with a certain restriction enzyme that characterizes the difference between two target sequences.

Polymerase Chain Reaction in Veterinary Molecular Diagnostics

Because of its predominance in research and diagnostic applica­tions, PCR assays will be discussed in more detail in later chapters. To give practitioners guidance about what to look for when PCR-based molecular diagnostic assays are offered, we will discuss some aspects such as design guidelines, differ­ences between traditional and real-time PCR assays, sampling, controls, and interpretation of results in greater depth.

PCR testing in its pure form is a three-cycle event: dena­turation of double-stranded target (or, in later cycles, PCR products), annealing of target and primers, and extension of the DNA strand from the primer.

COMPARING REAL-TIME POLYMERASE CHAIN REACTION WITH TRADITIONAL POLYMERASE CHAIN REACTION TESTING FOR DIAGNOSTIC APPLICATIONS. Real-time PCR testing was introduced into the marketplace in 1996 and replaced the tedious gel electrophoresis step to detect PCR products after amplifica­tion. In one survey, 98% of equine veterinarians knew about PCR testing, and 79% of equine veterinarians at universities knew the difference between conventional and real-time PCR testing.⁵ During gel electrophoresis, the PCR products are separated by size and visualized using a dye (ethidium bromide) that intercalates with the double-stranded DNA. Real-time PCR detects the PCR products by using an internal probe that is labeled with two fluorescent dyes: a reporter dye and a quencher dye. The fluorescent activity of the reporter dye is absorbed (quenched) by the quencher dye. The probe binds to the PCR products between the two PCR primers. If the primers are extended by the DNA polymerase, the 5' nuclease activity of the DNA polymerase digests the internal probe (hence hydrolysis probe) and releases the quenched fluorescence. Alternatively, hybridization probes that are not digested during amplification and that allow melting curve analysis can be used. This single change in the protocol regarding how to detect the PCR products revolutionized the use of PCR testing for both research and diagnostic applications. Most of the advantages resulting from this principle originate from the fact that the PCR tube does not have to be opened after a PCR assay for analysis. This principle is called closed-tube detection. The advantages are as follows:

1. Because of the closed-tube detection format, PCR products cannot escape from the PCR reaction containers. If escape occurs, it leads to the contamination of the PCR laboratory and subsequently the next PCR reactions. The consequence is false-positive PCR results. Real-time PCR efficiently eliminates this risk of PCR product carryover. There is also a second safety system called AmpErase UNG (Invitrogen, Grand Island, N.Y.),⁶ which eliminates contaminating PCR products.

2. Real-time PCR is a kinetic PCR principle, underlining the fact that PCR product accumulation is measured during every single PCR cycle in real time. Because of this fact, real-time PCR is a quantitative method.

3. The real-time PCR platform introduced in 1996 consisted of a laser-based thermocycler, software to design real-time PCR assays, reagents, disposables, and protocols. Because of its integrated design, PCR assays generated with the new system were highly standardized.

4. Data analysis is run on an attached computer that collects fluorescent data points. This principle eliminates all labora­tory steps after the real-time PCR cycling is finished. Because of the computer-formatted end analysis, results can be fed directly into the information and management systems of diagnostic laboratories. Real-time PCR has also enabled rapid cycling to finish the PCR process in less than 1 hour. Real-time PCR results therefore can be turned around significantly faster than with traditional PCR testing (normally within a 24-hour period).

5. Because of the probe used to detect the PCR products, real-time PCR is also a more specific assay than single-round traditional PCR. Only specific PCR products are detected by the probe. Real-time PCR testing, essentially a liquid-based hybridization method, is considered as specific as Southern blotting, which is a solid-based hybridization method.

6. Because real-time PCR incorporates hot-start enzymes, the specificity is even greater than with regular non-hot-start DNA polymerases. Hot-start enzymes are inactive at ambient temperatures and have to be heated to 95° C for 20 seconds to 10 minutes in order for the DNA polymerase activity to begin. Because of the hot-start nature, nonspecific binding of primers does not lead to unspecific PCR products and results in an overall increased specificity of the assay.

7. In combination, real-time PCR is described to be as sensitive as or more sensitive than double-round (nested) traditional PCR. Analytic sensitivity and limit of detection using real­time PCR testing is normally in the single molecule range, whereas the limit of quantitation with traditional PCR testing is in the range of 10 to 20 molecules.

Preanalytic Variables

In general, molecular diagnostic laboratories provide strict recommendations for sample collection, including shipping instructions. These instructions include specimen type, volume, and anticoagulant and specimen transport, storage, and handling. The sample type is largely influenced by the pathogenesis of the disease and plays a key role in the performance and interpretation of the test results. Veterinarians are advised to adhere to these recommendations, because the quality of the result is directly correlated to the quality of the sample and preservation of the nucleic acid content. Molecular assays often offer the convenience of using a small specimen acquired with a minimally invasive procedure. The diagnosis of herpesviruses is a classic example in which culture and neutralization assays have been largely replaced by PCR testing on a small volume of aspirate or swabs from mucosal surfaces. Molecular tests can detect the presence of small numbers of organisms, and the probability of detection increases when a larger volume of specimen is added to the amplification reaction. Because molecular assays do not need viable organisms for testing, more flexibility in specimen transport is possible than with culture methods.

Appropriate specimen collection and transport conditions are important to ensure successful extraction of intact nucleic acid and to prevent cross-contamination. Specimen transport and storage conditions are likely to vary among specimen types and between RNA and DNA tests. RNA is more susceptible to degradation, but in general molecular diagnostic samples should be cooled in order to slow the nucleic acid degradation within the sample. Because of the difficulty of maintaining a freeze chain, molecular diagnostic samples should only be cooled throughout the collection, storage, and transportation process in order to minimize sample deterioration. Detailed storage and shipping instructions are crucial for a successful molecular diagnostic workup. Practitioners should be aware of these recommendations and consider using appropriate cooling containers when samples are collected in the field. Such samples maintain stability if stored appropriately in a cooled environment. Freezing and in particular the thawing process often adversely affect the quality and should be avoided if not otherwise instructed by the laboratory.

Sampling errors are among the many preanalytic variables that can affect the outcome of the results. It is therefore recom­mended that the laboratory have the appropriate labeling material available for blood containers or other sample types. Blood or body liquid contamination on the outside of the containers is an obvious cause of sample cross-contamination before the samples are manipulated in the laboratory. Proper collection procedures are essential to prevent these kinds of artifacts.

NUCLEIC ACID EXTRACTION. Many commercially available manual and automated methods have been successfully applied to infectious disease testing using a variety of clinical materials. Especially in veterinary molecular diagnostics, the variety of sample types can be challenging for processing through a single platform but can be achieved by validating sample type-specific pretreatment procedures in order to create a lysate of similar viscosity and nucleic acid concentration, which maximizes the extraction efficiency. Automated or semiautomated platforms are rapid and usually require only a small volume of specimen. In general, these systems are total nucleic acid extraction systems and support the parallel analysis of DNA and RNA pathogens from the same sample in a panel configuration. In addition, commercially available automated systems can reduce hands-on labor requirements and speed up the process significantly, leading to even shorter turnaround times. Most systems can accommodate sample volumes ranging from 100 to 500 μL and even 1 mL of specimen lysate. Nucleic acid binding capacity varies and can be as high as 200 μg per individual extraction position, which allows the maintenance of a good limit of detection even when many infectious agent targets are analyzed in parallel for a given panel.

INTERPRETATION OF RESULTS. Interpretation of results obtained with molecular assays for infectious diseases requires understanding of the pathogenesis and biology of the target organisms. Some challenges are unique to molecular tests and are different from considerations in interpreting other micro­biological tests. Such differences are related to the distinction of viable from nonviable organisms and the correlation of nucleic acid detection with presence of disease or disease association.

Interpretation of a negative result includes information about the sensitivity and nucleic acid extraction efficiency. A false­negative result may be caused by a degraded or unstable sample. Insufficient volume or inappropriate sample type, inadequate sampling procedures, and transport problems are additional sources for potentially false-negative results. Sample-specific internal positive controls targeting endogenous genes such as the universal 18S rRNA (ssrRNA) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene help to identify these problems. These so-called internal positive controls allow quantification of the sample-specific nucleic acid (both DNA and RNA) and allow validation of a diagnostic sample for its use in PCR; samples below a certain quantity of DNA are at risk of being called false negative; therefore repeat collection can be recom­mended. In addition, inhibition phenomena originating from difficult sample matrixes such as feces, urine, or environmental samples contaminated with trace amounts of soil (humic acid) have to be controlled with internal positive controls to assess the inhibitory effects.

The factors requiring consideration for the interpretation of positive results include assay specificity and contamination issues. PCR testing or any other target amplification method is subject to these considerations. Real-time PCR testing using closed-tube detection procedures reduces the risk of PCR product carryover as a source of false-positive results and the availability of systems, which mark the PCR products when produced with certain types of nucleotides (uracil instead of thymidine), allows the specific degradation of contaminating PCR products before the next PCR amplification occurs (AmpErase UNG system). Combined with air movement control in the molecular diagnostics laboratory (overpressurized clean laboratory and negative-pressurized sample preparation labora­tory) and limited access to trained laboratory technicians, great safety can be achieved, leading to valid results.

In general, molecular assays do not provide information about the viability of an infectious agent. Exceptions to this are DNA viruses, bacteria, and parasites analyzed for transcribed genes instead of their genomic DNA. This approach allows the assessment of the replication competence of a target and therefore is highly correlated with viability. Examples are toxin quantification, differentiation of vaccination versus infection, and herpesvirus quantification in order to differentiate between lytic (acute) and chronic (latent) infection, which has immediate implications to the ability of the virus to cause or contribute to clinical signs. In addition, spliced RNA occurs only if viral genes are actively transcribed and provides a means for obtaining information about the replication activity of a virus and the stage of infection. In other cases, targeting the ribosomal RNA of parasites such as Toxoplasma and Cryptosporidium is a means for obtaining viability information and also may increase the analytic sensitivity.⁷

Detection of the nucleic acid of a pathogen does not neces­sarily ensure that the organism is the cause of the disease. A primary example is herpesvirus infections (EHV-1 and EHV-4), in which the detection of DNA may indicate the presence of lytic, nonreplicating, or latent virus. Studies indicated that high viral loads of EHV-4 DNA allow the formulation of a cutoff value to differentiate between lytic and latent infection.⁸ In this particular case, high viral loads were associated with the presence of clinical signs and viral RNA transcripts. Therefore quantitative real-time PCR testing can provide a means to obtain much improved disease association and invalidates the notion that PCR “just detects dead DNA.”

REPORTING OF MOLECULAR RESULTS. Reporting results for qualitative assays in infectious disease monitoring is simple: a sample either does or does not contain the nucleic acid of a target organism. Further relevant information includes the nucleic acid extraction efficiency, nucleic acid stability, and sample integrity determined with the various quality controls.

Reporting results for quantitative molecular infections is more complex. For veterinary medicine, some quantitative applications have been established and are progressively phased into diagnostic routine analysis. Such quantitative PCR (qPCR) tests combined with fully standardized protocols will continue to expand within the veterinary diagnostic market as our understanding of the biology and pathology of infectious agents grows.

Regulatory Considerations of

Molecular Laboratories

Veterinary molecular diagnostics is an emerging market with a growing level of regulation. Standards such as those defined by the American Association of Veterinary Laboratory Diag­nosticians (http://www.aavld.org), Molecular Methods (CLSI; http://clsi.org/), or MIQE (minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments) are adopted by commercial laboratories aiming to provide a comprehensive diagnostic service. Other sources for guidelines are U.S. Food and Drug Administration guidelines (www.fda.gov) for the detection of nucleic acids; ASTM guidelines (Standard Guide for Detection of Nucleic Acid Sequences by the Polymerase Chain Reaction Technique; www.astm.org); and the Association for Molecular Pathology (Recommendations for In-House Develop­ment and Operation of Molecular Diagnostic Tests; www.ampweb.org).

Guidelines for Clinicians to Select Molecular Diagnostic Laboratories

Veterinarians can use a variety of guidelines to select laboratories for molecular diagnostics. However, there are some major factors worth considering before samples are submitted. First, it is worthwhile to obtain information about the nature of the PCR test (traditional vs. real-time). Second, questions addressing the quality control and quality assurance system within a particular laboratory should be asked. Third, turnaround time, pricing, and the level of guidance with result interpretation and availability of consulting resources are additional factors worth investigating before samples are submitted.

Summary

As with any methodologies, qPCR is a work in continuous progress, development, and improvement. Although all of the methods described in this review use the Nobel Prize-awarded PCR process, the advantages and disadvantages of subtle differences in assay format, accuracy, and reliability of quantita­tion have to be rigorously compared. The next generation of quantitative real-time PCR processes will be increasingly automated, standardized, and miniaturized. The time for nucleic acid preparation and PCR amplification will be further reduced by using small microtiter-formatted microfluidic cards or silicon-based chip technology.^9-11 Amplification time has been brought down to several minutes by using an advanced nucleic acid analyzer (ANAA) consisting of a battery-powered array of silicon-based PCR microchips with thin-filmed resistive heaters, thus enabling ultrafast amplification, further pushing the technologic possibilities in order to improve the diagnostic results output.¹⁰

How will these new systems be incorporated in routine veterinary diagnostics? Veterinary medicine will experience a broad adoption of many quantitative assays for infectious agents and genetic abnormalities in the near future. The ability to standardize assays will allow high-throughput applications that have not been possible with traditional molecular methods. For this reason, applications for veterinary medicine may experience not only quantitative but also qualitative growth. These assays will help improve patient management and client satisfaction.