Diagnosing Infectious Diseases in the 21st Century--The Role of Molecular Biology
Susan E. Shaw, BVSc(Hons), MSc, DACVIM, DECVIM-CA, FACVSc, MRCVS
There has been a revolution over the past 20 years in our ability to dissect, analyse and understand the genome of microbial pathogens. These advances have been rapidly transferred and integrated into the diagnosis of veterinary pathogens from viral organisms to nematodes.
The genome of a pathogen contains all the information required to produce the proteins and RNA molecules required to successfully reproduce. The DNA sequences of the genes that are essential for life are quire similar between apparently unrelated organisms. However, the small subtle changes in DNA sequences that occur due to random mutations and which offer selective advantage to the organism or remain conserved over time, allow differentiation of one pathogen from another. All related organisms share similar DNA sequences and the more closely related they are, the more highly conserved the nucleotide is the sequence of their genomes.
DNA provides an excellent template on which to base diagnostic tests due to its stability and unique structure. Molecular technologies exist for extracting nucleic acids from all classes of pathogen in a variety of different sample types including blood, body fluids and tissues. This extracted DNA can then be used in a variety of assays that use a combination of base pairing characteristics and DNA repair enzymes from hyper-thermophilic bacteria to generate a diagnostic signal.
The Polymerase Chain Reaction (PCR)
The PCR has become the best known and most successfully implemented of the molecular technologies because of its application to rapid diagnostic testing for infectious diseases in a commercial context. It has the following inherent advantages.
1. It is a highly sensitive and specific technique for identification of minute quantities of microbial DNA in a diagnostic sample.
2. PCR can be performed on a wide variety of body fluids and tissues, multiple organisms can be detected simultaneously, and testing is independent of the mammalian species from which the sample is derived.
3. PCR will detect early infections, in many cases before organisms can be detected by traditional microscopic or microbiological methods and well before sero-conversion.
4. PCR detects active infection, not just prior exposure.
5. It can detect organisms that are difficult or impossible to culture.
6. It can be used where microbiological culture or organism detection is inadequate, time-consuming, or expensive.
7. PCR is invaluable where microbiological culture is hazardous to laboratory staff.
There are inherent technical limitations of PCR technology, but these are reduced in laboratories that use standardised protocols, conduct rigid validation and adhere to quality control procedures. The most important problem associated with PCR assays is that of DNA contamination either during sample collection or in the laboratory producing false positive results. In the laboratory, increased automation and the replacement of conventional testing (particularly those which greatly increase the amount of PCR product i.e., "nested" PCRs) with probe-based real time PCR, have gone a long way to controlling this problem.
False negative results can also occur if inhibitors to the PCR reaction are present such as residual haemoglobin or excessive amounts of PCR. These are uncommon if appropriate quality control is present and tests are completely validated.
Negative PCR results in clinical cases may be the result of submission of samples with degraded or no DNA. This is a problem with submission of blood samples for detection of organisms that sequestrate in other tissues (e.g., blood samples for Borrelia or Leishmania) or submission of poor quality swab samples with few cells ( e.g., conjunctival swabs for Chlamydophila).
Perhaps one of the most important aspects of PCR assay evaluation is its interpretation in clinical disease. Depending on the pathogen involved, a positive PCR result may not correlate with disease as the highly sensitive assays may detect very low numbers of organisms. However, detection of a "carrier-state" infection, may be advantageous in the overall management of inter-current disease.
The Conventional PCR Technique
PCR is an enzymatic procedure that allows almost unlimited amplification of specific nucleic acid sequences in vitro. During the process, the double stranded target DNA is multiplied in repetitive cycles of heating and cooling. During heating, the double helix of DNA is "unzipped". The resulting single strands can be used as targets for oligonucleotide probes (primers) which are carefully designed to have sequences exactly complementary to a portion of the target organism DNA. They key to the process of amplification is the addition of a thermostable DNA polymerase (such as that derived from the hot spring bacterium Thermus aquaticus) that remains active after numerous cycles of heating to 94°C. Newly synthesised double stranded DNA can thus be disassociated to act as templates for subsequent rounds of primer binding and DNA synthesis. The DNA product is amplified to a level at which it can be detected on a nitrocellulose gel system.
As the technology involved in determining nucleotide sequences of macro- and micro-organisms has progressed exponentially, there is now a vast array of sequences in databases such as Genbank. This information can be used to design novel primers using areas of the genome that are conserved between species, genera and families of pathogenic organisms or areas that are specific to individual strains. Consequently there has been an explosion in the commercial synthesis of oligonucleotide probes, and the generation of rapid DNA extraction kits, all of which have made PCR diagnosis commercially viable and easily accessible to veterinary practitioners.
The Advent of Real Time PCR Technology
In 1996, improvements in linking PCR and fluorescent signal detection resulted in the expansion of the technology referred to as "real-time" PCR. The PCR reaction can now be visualised as it occurs and the requirement for gel detection of the DNA product has been removed. In this method, fluorescent dyes are incorporated into the reporter nucleotide probes and included in the PCR reaction mixture. As the PCR product is produced, the amount of fluorescence increases proportionately. This is monitored throughout the assay and the data converted into quantitative results. This not only detects the presence of target DNA but allows measurement of the amount of DNA for assessment of pathogen load. This technology has the following advantages over conventional PCR methods.
1. Real time PCR is more rapid as the gel detection step is removed.
2. It is more sensitive.
3. As it allows quantitation, response to therapy can be monitored.
4. As it allows quantitation, the contribution of the target organism to the disease picture in an individual animal can be better interpreted.
The major disadvantage of real-time PCR at present is the expense of the thermocyclers and the probes. However, this can be expected to decrease in the near future. Already the technology has been miniaturised and converted into bed-side or crush-side kits. Most recently the technology has been introduced into hospitals for rapid MRSA screening and on the veterinary side into rapid on-site screening for avian influenza.
Examples of Use in Companion Animals
There are increasing numbers of PCR tests available for companion animal infectious diseases. Three of these will be illustrated using case examples: leishmaniasis, haemoplasmosis and granulocytic ehrlichiosis.
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