Molecular diagnostics: understanding assays for infectious diseases (Proceedings)

Molecular diagnostics have quickly become a mainstay in veterinary medicine, particularly in the area of infectious diseases. With the rapid advancement of technology, it is difficult to keep up with what is available as well as to understand what the results mean. Without an appreciation of the benefits and pitfalls of molecular technology for infectious disease diagnostics, over-interpretation of results can easily occur. The majority of molecular assays used for diagnosis of infectious diseases detect the pathogen itself, as opposed to serologic assays for pathogen-specific antibodies. These assays vary in sensitivity, specificity, turn-around-time and cost.

In the past, the most common methods for detection of pathogens in biologic samples involved use of antibodies specific for the antigenic epitopes of the agent, which are usually protein components. Immunofluorescence, ELISAs, and immunohistochemistry, for example, all utilize pathogen-specific antibody for detection of the agent in the sample submitted (e.g. serum, whole blood, tissue). Most of the newer molecular assays instead involve detection of the genetic material of the agent in question (DNA or RNA) rather than proteins.

The most common method currently for detecting genetic material of an infectious agent is polymerase chain reaction (PCR). With this technology, small pieces of an agent's genetic material are repeatedly copied, effectively amplifying it and allowing its detection. This methodology requires agent-specific targeting of the genetic material, thus most are relatively specific assays. For PCR, nucleic acid is extracted from the biologic sample submitted. The specific microbe, if present in the sample, is targeted using small pieces of DNA, termed primers, that "match" a genetic region of the microbe's genome. This targeted genetic region is then repeatedly copied using a specialized enzyme, termed Taq polymerase, named for Thermus aquaticus, the bacteria from which this enzyme was originally isolated. These bacteria live in the hot springs of areas like Yellowstone Park; because of their normal high ambient temperature, their enzymes are remarkably stable at high temperatures used in the this process. For all microbes, regardless of identity, the final product produced by the enzyme is double stranded DNA. Agents whose genome is RNA rather than DNA, as is the case for many viruses like canine distemper virus, FIV, feline coronavirus, and rabies virus, the genetic target must first undergo reverse transcription to produce a DNA copy of the RNA target. From this point, the amplification is the same as for agents whose genome is DNA.

Once amplified, the DNA product must be detected and its identity confirmed as that of the agent of interest. This can be done through a variety of methods, including size of the product following gel electrophoresis, dyes that bind to double stranded DNA, or use of labeled probes specific for the microbe that "light up" the DNA product.

Real-time PCR is a variation of conventional PCR in which the amplification process and DNA production can be followed in "real-time". Generally, it has increased sensitivity and specificity as compared to conventional PCR, and is also more rapid. With real-time assays, detection of the amplified nucleic acid is computer based rather than relying on visualization of the product. This may be done by a variety of methods but all involve fluorescence that is detected by a fluorimeter. One method uses a dye that binds only to double stranded DNA, while many, referred to as TaqMan assays, use a microbe-specific fluorescent-labeled probe. This latter methodology enhances the specificity of the assay.

With real-time PCR, amplification and detection all occur in the same tube, eliminating chance of contamination through post-PCR steps. Another advantage of real-time PCR is its ability to quantitate the amount of starting material. This quantitation may be absolute (precise value) or relative (estimation of amount of starting material). The cycle threshold number is used in this evaluation, and is the point in amplification when product fluorescence rises above background fluorescence. This value is indirectly proportional to the amount of starting genetic material; i.e. the higher the Ct value, the lower the amount of starting material – it takes more cycles to reach threshold with less starting material. Thus, a RT-PCR positive sample with a Ct of 20 (meaning it took 20 rounds of amplification to reach threshold) has more of the agent in the starting material, the sample submitted, than a sample that gives a Ct value of 40 (took 40 rounds of amplification to reach threshold). This can aid interpretation of positive results.

Often, a single agent is detected in a separate PCR assay. Some PCR assays are termed multiplex, which means that multiple agents are specifically targeted in a single assay. For example, a multiplex assay for feline herpesvirus and calicivirus can detect both viruses specifically in a single sample. In this situation, the probes for FHV and FCV are labeled with different fluorescent probes allowing distinction of the products.

PCR assays may be done on a variety of biologic samples, depending on the assay. For example, for canine parvovirus, feces can be used; for upper respiratory tract infection, swabs taken from the pharyngeal or nasal area may be used; and for FIV, blood is used. While the agent does not have to remain viable in the sample during transport from clinic to laboratory, the stability of the genetic material can vary among pathogens, often necessitating chilling during shipping.

One key aspect of PCR to remember is that it can be exquisitely sensitive, which is a double-edged sword. Very small amounts of genetic material are amplified repeatedly, leading to millions of copies. Because of this, subclinical, even latent infections can be detected. In addition, for agents included in vaccines, vaccinal virus can be detected for a short period following vaccination. This can be an issue in shelters where animals are vaccinated on intake and then break with disease within a few days after entry. Contamination can also be a problem, leading to false positive results. Stringent controls and experienced personnel are required for accurate results. In all cases, positive results must be interpreted in light of the other clinical parameters.

False negative results can occur as a consequence of viral genetic variation. The assay uses gene sequence-specific priming for the DNA synthesis; this priming is based on the nucleotide sequence of the target agent. With pathogens whose genetic sequence varies significantly among strains, this may lead to lack of DNA synthesis and amplification, and false negative results. This is a problem, for example, with FIV in cats where significant genetic variability exists. PCR has been used in an attempt to identify infection in vaccinated cats (for whom the antibody ELISA is ineffective); however, because of the significant strain variation of different FIV isolates, PCR is often negative, even in infected cats. Thus, as with any diagnostic assay, the results must be interpreted carefully.

Undoubtedly, the technology for diagnostics will continue to advance. Newer molecular techniques such as microarrays are already on the horizon. While these new techniques can enhance our ability to diagnose disease, one must always understand the factors, even limitations that impact the assay results.

References

Belak, S., et al., Advances in viral disease diagnostic and molecular epidemiological technologies. Expert Review of Molecular Diagnostics, 2009. 9(4): p. 367-381.

Brown, M.A., et al., Genetics and Pathogenesis of Feline Infectious Peritonitis Virus. Emerging Infectious Diseases, 2009. 15(9): p. 1445-1452.

Decaro, N., et al., Genetic analysis of canine parvovirus type 2c. Virology, 2009. 385(1): p. 5-10.

Dowers, K.L., et al., Association of Bartonella species, feline calicivirus, and feline herpesvirus 1 infection with gingivostomatitis in cats. Journal of Feline Medicine and Surgery, 2010. 12(4): p. 314-321.

Guptill, L., Feline Bartonellosis. Veterinary Clinics of North America-Small Animal Practice, 2010. 40(6): p. 1073-+.

Gut, M., et al., One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses. Journal of Virological Methods, 1999. 77(1): p. 37-46.

Hoffmann, B., et al., A review of RT-PCR technologies used in veterinary virology and disease control: Sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health. Veterinary Microbiology, 2009. 139(1-2): p. 1-23.

Meurs, K.M., et al., Molecular screening by polymerase chain reaction detects panleukopenia virus DNA in formalin-fixed hearts from cats with idiopathic cardiomyopathy and myocarditis. Cardiovascular Pathology, 2000. 9(2): p. 119-126.

Ryan, M.T. and T. Sweeney, Integrating molecular biology into the veterinary curriculum. Journal of Veterinary Medical Education, 2007. 34(5): p. 658-673.

Sjodahl-Essen, T., et al., Evaluation of different sampling methods and results of real-time PCR for detection of feline herpes virus-1, Chlamydophila felis and Mycoplasma felis in cats. Veterinary Ophthalmology, 2008. 11(6): p. 375-380.

Sykes, J.E., Feline hemotropic mycoplasmas. Journal of Veterinary Emergency and Critical Care, 2010. 20(1): p. 62-69.

Westermeyer, H.D., H. Kado-Fong, and D.J. Maggs, Effects of sampling instrument and processing technique on DNA yield and detection rate for feline herpesvirus-1 via polymerase chain reaction assay. American Journal of Veterinary Research, 2008. 69(6): p. 811-817.