Diagnostic testing is an important tool that veterinarians use to understand the cause and seriousness of a disease. Traditionally, this involved a trade-off between speed or accuracy, but improvements in qPCR testing are bringing point-of-care molecular diagnostics into practical use.
Traditional Diagnostic Testing: Point-of-Care vs. Outsourcing
Diagnostic tests fit into two categories: tests that are carried out at the veterinary clinic at the point-of-care (POC) or tests carried out at remote sites such as a reference laboratory.
Typically, tests performed at a laboratory are more complex and need experienced technicians to perform them. On the other hand, POC tests are simple, easy to perform, and less likely to generate erroneous results. They are also more amenable to automation.
Since POC tests can be performed at the clinic itself, they have a faster turnaround time than other tests. This has improved the standard of care in companion animal medicine. However, diagnostic tests for infectious diseases rely on reference laboratories for a definitive diagnosis. This, coupled with the sub-optimal performance of available POC tests based on microscopy, serology, or antigen detection, leaves the veterinarian with inadequate information for developing an evidence-based treatment plan.
Neither option was completely satisfactory, but there were no other practical options available until recently.
qPCR Applications in Veterinary Medicine
Real-time Polymerase Chain Reaction (qPCR) is the gold standard molecular diagnostic tool because of its speed and accuracy compared with traditional diagnostic tests. qPCR provides quantitative data on the analyte by identifying the point during cycling the product was first detected through real-time monitoring of product accumulation.
qPCR applications in veterinary medicine can be divided into three areas:
- Detection of pathogens causing infectious diseases (e.g., detection of bacteria, virus, fungi, and protozoans causing diseases in animals)
- Gene expression analysis (e.g., gene expression changes associated with disease conditions including cancer)
- Allelic discrimination (e.g., identification of genetic mutations affecting diseases, drug reactions, etc.)
Until very recently, qPCR was confined to testing at reference laboratories, due to constraints imposed by manual nucleic acid extraction, contamination control, and the extensive training required to either execute the test or interpret the data.
Pathogen detection tests are the most widely performed qPCR tests in veterinary patients, as a rapid diagnosis can favorably impact treatment outcomes.
Still, the practical effectiveness of these tests was offset by cost and turnaround time. Shipping samples to a reference lab meant veterinarians had to wait days for results, eliminating qPCR as a realistic option in many cases.
Sample-to-answer qPCR instrumentation — with integrated sample preparation and automated result interpretation and reporting in the clinic — offers significant improvement to diagnosis and treatment of infectious diseases in companion animals.
qPCR in Companion Animal Medicine at Point-of-Care
qPCR has multiple applications for infectious disease diagnosis in the companion animal clinic, based on the type of pathogen or disease conditions targeted.
Detecting Viral Pathogens During the Clinic Visit
The gold standard reference method for diagnosing viral infections is to isolate and culture the virus for identification. This method is laborious and requires mammalian cell cultures, a dedicated facility, and trained personnel, which makes it difficult even for most reference laboratories.
qPCR is uniquely suited for virus detection directly from samples in the clinic because of the shorter turnaround time and ease of use; nearly anyone qualified to work in a veterinary clinic can prepare the required samples, run the test, and get actionable results. Some of the most common viral pathogens affecting cats and dogs that qPCR could detect include, but are not limited to, the canine distemper virus, canine influenza virus, canine parvovirus, and feline infectious peritonitis virus.
Detecting Difficult-to-Culture Bacterial Pathogens
Slow growing or fastidious bacterial pathogens are responsible for a variety of clinical conditions that bring cats and dogs into the clinic. Some examples include Mycoplasma spp., Chlamydophila spp., Leptospira spp., and tick-borne pathogens such as Borrelia spp., Ehrlichia spp. and Anaplasma spp.
Routine diagnostic procedures, such as immunofluorescence tests (Chlamydophila spp.) and serology (Leptospira spp. and tick-borne pathogens), suffer from suboptimal sensitivity and specificity. Widespread vaccination of dogs against Leptospira spp. further complicates the interpretation of serological test results.
The high sensitivity of qPCR allows for rapid detection of pathogen-specific nucleic acids and are superior to serology, especially in acutely ill patients, as results are obtained well before seroconversion. qPCR can also identify cases of active infection by differentiating seropositive animals due to vaccination from actual infection.
Detecting Pathogen-Associated Antimicrobial Resistance (AMR) Markers
Antimicrobial sensitivity profiling of bacterial pathogens through determination of minimum inhibitory concentrations (MIC) of various antibiotics is critical to clinical decision making. Traditionally, it was the best way to determine whether the pathogen at fault was susceptible to treatment with a specific antimicrobial agent.
However, the multi-day turnaround time of standard antimicrobial susceptibility testing (AST) remained a bottleneck to evidence-based treatment decisions in the clinic. Veterinarians could often begin treatment with one agent, only to find they had made an inappropriate choice when test results finally came back.
POC qPCR offers a promising alternative by testing directly for the AMR genes that are predictive of resistance phenotypes in bacterial pathogens. Some examples of AMR genes that can be detected directly from clinical samples include mecA in methicillin-resistant Staphylococcus pseudintermedius, carbapenemases (KPC, NDM, IMP, VIM, OXA-48) in Enterobacteriaceae, tet (tetracycline resistance), and sul (sulfonamide resistance).
Information on the presence or absence of specific AMR markers allows veterinarians to narrow down their treatment options and make evidence-based treatment decisions.
Using Syndromic Panels to Identify Disease-causing Pathogens
Syndromic testing is when one sample is used to test for multiple possible pathogens and co-infections at once, based on the symptoms an animal presents in the clinic. This allows veterinarians to identify the causative agent and an appropriate method of treatment from a single test.
Incorporating assays which detect AMR markers for first- and second-line therapeutics can provide the veterinarian with data on the pathogens involved and their predicted resistance profile, as well. This enables them to select an effective antimicrobial agent at diagnosis.
Some examples for syndromic qPCR panels with most diagnostic value in the veterinary clinic are as below:
Urinary tract infection (UTI) Panels
Bacterial UTI is a common clinical condition and a major reason for antimicrobial use in cats and dogs, with approximately 14% of all dogs having at least one instance of UTI in their lifetime.
A handful of pathogens such as Escherichia coli, Staphylococcus spp., Proteus spp., Enterobacter spp., Enterococcus spp., Klebsiella spp., Pseudomonas spp., and Streptococcus spp. cause the great majority of UTI in cats and dogs. Amoxycillin and Sulfa-TMP are considered good first-line treatment options.
Therefore, a panel containing common bacterial pathogens and their associated AMR markers coding for beta-lactam, sulfonamide, and trimethoprim resistance is a good choice for diagnosis of UTI in companion animals and can provide faster results than culture and sensitivity testing (C&ST).
Skin and Soft tissue infection (SSTI) Panel
Diseases of the skin including pyoderma, otitis, wounds, and abscesses account for as much as 40–50% of antimicrobial prescriptions in cats and dogs.
The most common pathogens associated with skin infections in companion animals include Staphylococcus spp. such as S. pseudintermedius and S. schleiferi. These pathogens isolated from skin infections will likely carry the mecA gene and are often resistant to multiple classes of antimicrobials. Pseudomonas spp., Proteus spp., Streptococcus canis and other gram-negative pathogens are also isolated from skin infections.
Lincosamides, cephalosporins, amoxicillin-clavulanate, and Sulfa-TMP are all recommended as first-line therapy for bacterial folliculitis in dogs. A panel containing assays for detecting these pathogens and AMR markers encoding resistance to these first-line therapy agents and mecA would be a useful diagnostic aid for infection of skin and soft tissue in companion animals.
Respiratory tract infection (RTI) Panel
Viruses (e.g., canine adenovirus type 2, canine distemper virus, canine herpesvirus, canine parainfluenza virus, canine respiratory coronavirus, canine influenza virus, feline calicivirus, and feline herpesvirus) and bacterial pathogens (e.g., Bordetella bronchiseptica, Mycoplasma spp., Chlamydophila felis, and Streptococcus spp.) typically cause RTIs in cats and dogs.
Additionally, other bacteria such as Staphylococcus spp., Pasteurella spp., Enterococcus spp., and Pseudomonas spp. cause secondary infections following primary viral infections, or less commonly other conditions.
RTIs account for a significant proportion of cases necessitating antimicrobials use in cats (24%) and dogs (16%). Some of the first-line therapeutic agents recommended to treat bacterial RTIs include amoxicillin, doxycycline, and clindamycin. Therefore, a hybrid panel containing assays for viral and bacterial pathogens along with AMR markers coding for resistance to these agents would help in differential diagnosis of companion animal RTI and selection of appropriate treatment options.
Automated qPCR is a Game Changer in Companion Animal Medicine
An automated sample-to-answer qPCR system with integrated sample preparation, data analysis, and result interpretation and reporting provides veterinarians with high quality diagnostic test results for infectious diseases at the POC. Such a system provides valuable information on the etiology of the disease condition and molecular signatures of AMR profiles associated with the pathogen(s) detected.
This enables veterinarians to make evidence-based treatment decisions during or immediately after consultation, without having to wait for days to get traditional diagnostic test results. Avoiding the need for empirical decision making, which can often lead to unnecessary over-treatment or under-treatment, can promote antimicrobial stewardship and facilitate informed patient care.