Molecular Awareness and Testing Strategies

Many veterinarians are aware of the availability of molecular diagnostic tests and have used these techniques in their practice. However, the lack of a market dominator for molecular diagnostics and the relatively fragmented market leads to confusion. This confusion is mostly based on a lack of directed education within the veterinary community. Most veterinarians rely on continuing education offered at local or national meetings to improve their knowledge base of molecular diagnostics. As more and more practitioners use PCR to diagnose infectious diseases, an understanding of the involved processes is important. Further, the indications for using PCR and interpretation of results are often confusing and warrant more education within the veterinary community. The differences among laboratories in protocols used add to the confusion caused by the lack of an acceptable standard.

Parallel testing for multiple infectious agents in highly standardized platforms is a central component of molecular assays; it essentially allows several detections, for both DNA and RNA pathogen targets, to happen simultaneously on a single 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 enables efficient workup of complex clinical syndromes with general or nonspecific clinical signs. These clinical situations do not allow for easy diagnostic decision making by the veterinarian because multiple infectious agents can be responsible for a given clinical picture. Even though veterinarians tend to make a single-pathogen diagnosis, it has become more evident in recent years that many syndromes are caused by coinfections. Panel testing on a large scale will uncover unknown dual or triple infections in animals, which can diffuse the clinical picture. It has long been speculated that seemingly clinically irrelevant equine herpesvirus type 2 (EHV-2) infections in horses may actually aggravate and diffuse the clinical picture presented by secondary infections. More characteristic examples are known from companion animal respiratory infections, which are often initiated by a subclinical virus infection that leads the way to secondary infections.

Preanalytical Variables and Result Interpretation

In general, molecular diagnostic laboratories provide precise recommendations for sample collection and shipping. These instructions pertain to specimen type, volume, anticoagulant, transport specifications, storage, and handling. The sample type or types needed are largely influenced by the pathogenesis of the disease and play 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 quality of the sample and preservation of the nucleic acid content. Whole blood samples are collected aseptically into evacuated blood tubes containing EDTA; body fluids (e.g., thoracic, abdominal, joint, cerebrospinal, tracheal wash, bronchoalveolar, and guttural pouch lavage fluid) and tissues should be collected into serum tubes without additives; nasal or nasopharyngeal secretions should be collected with rayon- or Dacron-tipped swabs and are best kept in a serum or conical tube; fecal material should be collected into small fecal cups or serum tubes. All samples must be sent cooled on blue ice by express mail overnight to the laboratory. Freezing of samples should be avoided because of the detrimental effects of the thawing process on NA. Short-term storage for a period of 2 to 3 days before shipment (such as would be necessary over a weekend) should be done in a refrigerated compartment. Each sample should be properly labeled and accompanied by a submission form containing information on the animal, owner, veterinarian, sample, and suspected pathogens. Most submission forms can be downloaded from the respective laboratory’s website. The laboratory should be notified in advance, and inquiry should be made about the availability of the offered tests as well as the expected turnaround time and the associated costs. Incoming samples normally are processed the same day, and PCR results usually are available within 24 to 72 hours (including shipping) if the purified NA passes the internal sample quality controls (confirming proper collection, storage, shipping, and NA extraction) and other associated quality controls, such as PCR-positive and PCR-negative controls, internal positive control (to confirm absence of PCR inhibitors), and negative extraction control (to confirm absence of cross-contamination during the NA extraction process). Veterinarians should be aware of the quality controls run on their diagnostic samples by inquiring with the respective diagnostic laboratory.

Interpretation of results obtained with molecular assays for infectious diseases necessitates 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 microbiologic tests. Such differences are related to the distinction between viable and nonviable organisms and the correlation of NA detection with presence of disease or disease association.

Interpretation of a negative result requires taking into consideration information about the sensitivity of the PCR test, limit of detection, and the NA extraction efficiency as indicated by the use of quantitative internal sample controls. A false-negative result may be caused by a degraded or unstable sample. Insufficient or inappropriate sample type, inadequate sampling procedures, and transport problems are additional sources of false-negative results. Sample-specific internal positive sample controls targeting endogenous genes, such as the universal 18S rRNA (single-stranded rRNA) or the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, help to overcome this problem, particularly if the lab uses them in a quantitative fashion to assess the quantity of NA going into a PCR protocol, which directly influences the limit of detection of the assay. In addition, inhibition phenomena originating from sample matrixes, such as feces, urine, or environmental samples contaminated with soil or surface water, have to be controlled with internal positive controls to assess the inhibitory effects on the PCR process.

The factors to consider for interpretation of positive results include assay specificity and contamination issues. Polymerase chain reaction or any other target amplification method is subjected to these considerations. Real-time PCR with closed-tube detection procedures reduces the risk for PCR product carryover as a source of false-positive 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 that are analyzed for the presence of RNA molecules, such as rRNA and transcribed genes in the form of messenger RNA, instead of their genomic DNA equivalents. Targeting spliced RNA occurring at certain steps during the replication cycle of particular viruses provides additional information into the replication activity of a virus. In other cases, targeting the ribosomal RNA of parasites such as Toxoplasma spp and Cryptosporidium spp is a means of obtaining viability information and also may increase the analytical sensitivity.

Detection of a pathogen’s NA in a sample does not necessarily indicate that the organism is the cause of the disease. However, using the quantitative information of a real-time PCR result may give further insight and provide a means for evaluating disease association. Primary examples are herpesvirus infections (EHV-1 and EHV-4), in which the quantitative detection of DNA may indicate presence of lytic, nonreplicating, or latent virus. Studies have indicated that high viral loads of EHV-4 and EHV-1 DNA allow formulation of laboratory-specific cutoff values to differentiate between lytic and nonreplicating virus. In such cases, high viral loads are generally associated with the presence of clinical signs and the presence of viral RNA transcripts indicating virus replication. Therefore quantitative real-time PCR can provide a means of obtaining information about the disease association, a crucial criterion for the equine practitioner in making the correct diagnosis.

Veterinarians can use a variety of guidelines to select laboratories for molecular diagnostic testing. Certain questions are worth asking before samples are submitted to a molecular diagnostic laboratory. These questions should cover three areas. First, it is worthwhile to obtain information about the nature of the PCR testing platform (traditional versus real-time). Second, questions should be asked about the quality control and quality assurance system within a particular laboratory. In particular, it is useful to know whether whole processes are controlled or just single point controls are used, and how contamination is avoided and confirmed to be absent within the laboratory. Third, additional questions about turnaround time, pricing, and the level of guidance with result interpretation are worth asking before samples are submitted.