The polymerase chain reaction (PCR) technique was first described in 1985.1 This technique results in the cyclic amplification of a single strand of DNA to produce an exponential number of identical copies that then can be easily detected, usually on a gel (conventional or end-point PCR), to determine if it is the predicted size for the reaction (Fig. 41.1). The enzyme used in PCR can only duplicate strands of DNA. To detect RNA, the sample must first have a reverse transcription (RT) step to create a complementary strand of DNA from the target RNA. Amplification of the complementary DNA by PCR is then performed; this method is commonly known as RT-PCR. PCR is superior in sensitivity to probe hybridization techniques because of this amplification step. The great sensitivity of these assays requires strict adherence to good laboratory practice to avoid false-positive results from contamination.

PCR is frequently used in veterinary medicine to detect infectious disease agents. The primers used in PCR can be designed to amplify the nucleic acids only of members of a certain genus, species, or even strain of organism. The use of broad-range or degenerate primers amplifying members of an entire genus or even kingdom can be used, targeting highly conserved regions of the nucleic acids. The most common application of this is for rapid detection and identification of eubacteria or fungi in clinical samples.2,3 Subsequent analysis of the PCR product may then be used to identify the infecting organism much more rapidly than traditional microbiologic techniques and may be more sensitive for detection of fastidious organisms. However, diagnostic PCR assay results do not provide antimicrobial sensitivity results and so for organisms that can be grown, PCR is complementary to traditional culture techniques. But the use of PCR for amplification of certain genes that encode for antimicrobial resistance is starting to gain clinical use as well and may provide additional rapid information before sensitivity results are available.⁴

When DNA from a single organism is targeted in an assay, it is termed a singleplex PCR assay. If multiple targets are to be amplified in a single assay, it is termed a multiplex PCR assay. While it is clearly most attractive to investigate the presence of multiple organisms in a sample using one PCR assay, each target sequence competes with the others for the enzyme, nucleotide, and various buffers and ions that allow the reaction to proceed. Therefore, multiplex assays can be less sensitive than singleplex assays.

It is difficult to acquire quantification information using traditional end-point PCR. Real-time PCR or quantitative PCR (qPCR) is the most recent application of PCR.⁵ Digital PCR is the newest form of this technology which fractionates a DNA sample into 20,000 droplets and amplifies the target in each droplet (see Cytauxzoon felis section). With qPCR, production of DNA is monitored during each amplification cycle so that the original starting quantity could be extrapolated by identification of the logarithmic amplification phase of each individual reaction. This technique uses fluorescent dyes or probes that produce a signal after formation of the product (Fig. 41.2). During each amplification cycle, a detector records the amount of fluorescence in the sample. Pathogen detection and load are one of the many applications of this technology. This assay has all the advantages of traditional end-point PCR (good sensitivity and specificity) and offers a more rapid result and the ability to quantify microbial DNA or RNA load and so can be used to monitor therapy in some cases (see the following sections of the chapter). Because qPCR is very sensitive, strict quality control must be maintained. In addition, accuracy of quantification is reliant upon the availability of a reproducible, high-quality, standard curve. Although minimum laboratory standards have been proposed and are generally met for published protocols,⁶ many diagnostic laboratories use proprietary reactions that are not subject to peer review. Thus, all laboratories providing PCR assays may not be equivalent; using laboratories that have published results of their assays may be prudent.

Feline calicivirus (FCV) is a common differential diagnosis for cats with clinical evidence of rhinitis and stomatitis. Less commonly, FCV is associated with conjunctivitis, polyarthritis, and lower airway disease in kittens. Virus isolation can be used to document current infection but takes at least several days for results. Because of widespread exposure and vaccination, the PPV of serologic tests is poor. Reverse transcriptase PCR assays can be used to amplify the RNA of FCV, and results can be returned quickly. However, these assays also amplify vaccine strains of FCV.7 This viral RNA can be amplified from samples collected from normal carrier cats as well as clinically ill cats and so RT-PCR has poor PPV for disease caused by FCV.⁸ For example, in one study in our laboratory, presence of FCV RNA failed to correlate with the presence or absence of stomatitis in cats.⁹ In addition, amplification of FCV RNA cannot be used alone to diagnose virulent systemic calicivirus infection. The NPV for FCV RT-PCR assays is currently unknown. Feline caliciviruses, as RNA viruses, have genetic variability among the different strains. Depending on the viral genetic region targeted by the assay, the degree of genetic variation among strains at that site will vary. Most laboratories design their assays to target conserved regions of the viral genome, but even this cannot guarantee that all strains are detectable by any individual assay.

Feline herpesvirus (FHV-1) is a common differential diagnosis for cats with clinical evidence of rhinitis, stomatitis, conjunctivitis, keratitis, and facial dermatitis. Because of widespread exposure and vaccination, the PPV of serologic tests is poor. The presence of FHV-1 can be documented by direct fluorescent staining of conjunctival scrapings, virus isolation, or PCR. Feline herpesvirus-1 DNA can be amplified from conjunctiva, nasal discharges, and the pharynx of healthy cats; and so, the PPV of conventional PCR assays is low.¹⁰ Currently used PCR assays also amplify vaccine strains of FHV-1, further decreasing the PPV of the assays.¹¹ In studies in our laboratory, presence of FHV-1 DNA failed to correlate with the presence or absence of stomatitis in cats and failed to predict a treatment response to cidofovir.^9,12

Quantitative PCR for FHV-1 was beneficial in documenting a treatment effect to cidofovir in one study of experimentally infected cats.¹³ However, attempts to correlate FHV-1 viral DNA load to clinical illness in field studies have had mixed results. In one study of cats with conjunctivitis, results of FHV-1 qPCR failed to correlate with the presence of conjunctivitis.¹⁴ In another study, FHV-1 qPCR assay results in cats with rhinitis could discriminate between healthy cats and cats with active rhinitis but not between cats with active rhinitis and cats that had recently recovered.¹⁵ The NPV of FHV-1 PCR assays is also in question because many cats that are likely to have FHV-1-associated disease are negative. This may relate to clearance of FHV-1 DNA from tissues by a hypersensitivity reaction. Tissue biopsies have greater sensitivity than conjunctival swabs but do not necessarily have greater predictive value. Feline herpesvirus-1 DNA can be amplified from aqueous humor of some cats, but whether this indicates FHV-1-associated uveitis is unknown.¹⁶

PCR assays have also been used to document the presence of nucleic acids of several influenza viruses as well as severe acute respiratory syndrome coronavirus 2 in samples from cats.^17,18 While less data are available for these agents, some of the same problems in interpretation of results that occur with FCV and FHV-1 exist.

Mycoplasma spp., Chlamydia, and Bordetella bronchiseptica are other common respiratory pathogens in cats. As for FHV-1 and FCV, positive PCR test results for these organisms cannot be used to distinguish a carrier from a clinically ill cat. A meta-analysis showed an association with disease in Mycoplasma spp. PCR-positive cats with upper respiratory tract disease, though the association was strongest in cats in a household situation compared to cats in a shelter situation.¹⁹ In another study, use of a qualitative end point Mycoplasma spp. PCR assay did not correlate with treatment responses to topical administration of a tetracycline.¹² In addition, diagnostic PCR assays do not provide antimicrobial drug susceptibility testing. For cats with potential bordetellosis, culture and sensitivity is the optimal diagnostic technique, especially if an outbreak is occurring. The bacterial microbiome of cats with and without nasal diseases was reported.²⁰ How to use this information most effectively in the management of clinical disease will be determined in future studies.

Toxoplasma gondii DNA has been amplified from airway washings of some cats with lower respiratory tract disease. Therefore, PCR is an option for evaluation of samples from diseased cats when the organism is not identified with cytology.

The diagnosis of Giardia spp. infection is generally made with the combination of fecal flotation techniques and wet mount examination. Fecal antigen tests are also accurate and when one commercially available assay was combined with fecal flotation, the combined sensitivity was 97.8%.21 Fecal PCR assays are often falsely negative for Giardia spp. DNA because of PCR inhibitors in stool. For example, one Giardia PCR assay had a 41% false negative rate compared with fecal flotation and direct fluorescent antibody staining when applied to canine fecal samples. Therefore, PCR should not be used as a screening assay for this agent.²² The best use for Giardia spp. PCR is to determine whether the infective species is a zoonotic assemblage. Currently, assemblage determination should be performed on more than one gene for most accurate results.²³

Although Cryptosporidium spp. infection is common, it is unusual to find Cryptosporidium felis oocysts using fecal flotation techniques in cats. Acid-fast staining of a thin fecal smear is insensitive. Antigen assays titrated for use with human feces are inaccurate when used with cat feces. Thus, PCR may aid in the diagnosis of cryptosporidiosis in dogs and cats and has been shown to be more sensitive than immunofluorescence assay (IFA) in cats.^24,25 Cryptosporidium spp. PCR assays are indicated in IFA-negative cats with unexplained small bowel diarrhea and when the genotype of Cryptosporidium must be determined. However, C. felis infection in cats is common; therefore, positive tests results do not always prove that the agent is the cause of the clinical disease. No drug is known to eliminate Cryptosporidium spp. infection, and small animal strains are not considered significant zoonotic agents; therefore, PCR is never indicated in healthy animals. PCR was used to amplify the DNA of another gastrointestinal pathogen, Cystoisospora felis, and was more sensitive than detection of oocysts by fecal flotation in one study.²⁶

Toxoplasma gondii is only shed for about 7 to 10 days, and millions of oocysts are generally shed during this time, making the organism very easy to identify. Thus, PCR assays are usually not needed to diagnosis this infection.

Trophozoites of Tritrichomonas foetus can be detected on wet mount examination of fresh feces and can be performed in-clinic. PCR for T. foetus DNA is indicated if wet mount examination is negative; results return more quickly than culture results. However, DNA of T. foetus can be detected in healthy carrier cats; and so, positive results do not always prove illness from the organism.²⁷ The method of collection may also influence results; feces collected by loop gave more sensitive results than colonic flush in one study.²⁸ Spontaneous and ronidazole-induced clearance has been noted.²⁹ Proving a kitten is negative for T. foetus DNA in feces before being moved to a naïve cattery is indicated.

DNA from bacterial pathogens can be amplified from the feces of cats with diarrhea.³⁰ While DNA of Salmonella spp. and Campylobacter spp. can be amplified, suspected salmonellosis or campylobacteriosis cases should be cultured rather than assessed by PCR to determine the antimicrobial susceptibility patterns. Whether PCR assays for Clostridium perfringens or toxin genes are important in the diagnosis of diarrhea in cats is relatively unknown.

PCR and genetic sequencing can be used to the define the fecal microbiome and can be used to follow treatment effects and define pathogenic mechanisms.^31,32 How best to use these techniques clinically will be determined in future studies.

PCR for parvovirus in feces is occasionally positive in kittens that are negative for antigen and PCR can be used to determine whether dog or cat strains are present.³³ However, it has been shown that cats vaccinated with modified live panleukopenia–containing vaccines shed parvovirus DNA in feces.³⁴ Thus parvovirus PCR testing should not be used to diagnose panleukopenia virus outbreaks in recently vaccinated cats.

Because virus isolation is not practical clinically, RT-PCR is used most frequently to detect coronavirus RNA in feces. However, positive test results do not differentiate FIP-inducing strains from enteric coronaviruses. Additionally, presence of coronavirus RNA does not always correlate with the presence of diarrhea.

Mycoplasma haemofelis (Mhf), “Candidatus Mycoplasma haemominutum” (Mhm), and “Candidatus M. turicensis” (Mtc) all can be found in cats. In experimentally infected cats, Mhf is apparently more pathogenic than Mhm and it appears that Mtc has intermediate pathogenicity. Diagnosis is based on demonstration of the organism on the surface of erythrocytes on examination of a thin blood film or with a PCR assay. Organism numbers fluctuate so blood film examination can be falsely negative up to 50% of the time. The organism may be difficult to find cytologically, particularly in the chronic phase. It has been known for many years that PCR assays are the tests of choice because of good sensitivity.35 Primers are available that can amplify all three hemoplasmas. Real-time PCR assays can be used to monitor copy numbers during and after treatment.³⁶ PCR assays should be considered in the evaluation of cats with unexplained fever or anemia that are cytologically negative for hemoplasma. In addition, the American College of Veterinary Internal Medicine recommends screening cats for use as blood donors by PCR assays for hemoplasmas.³⁷ Many cats (approximately 15%) are carriers of the relatively nonpathogenic Mhm so positive test results may not always correlate with the presence of disease (i.e., poor PPV).

Cats can be infected by an Ehrlichia canis-like organism³⁸ and Anaplasma phagocytophilum.^39,40 Little is known about Anaplasma platys, Ehrlichia ewingii, and Ehrlichia chaffeensis and associations with disease in cats. Because the organisms are in different genera, serologic cross reactivity is variable. Thus, although the clinical syndromes can be similar, there is no single serologic test to document infection, and there is currently no standardized serologic test for cats. In addition, some cats with E. canis infection do not seroconvert, so PCR assay is superior to serology in cats.³⁸ PCR assays can be designed to amplify each organism. Alternatively, primers are available to amplify all the organisms in a single reaction, and then sequencing can be used to determine the infective species. However, positive test results do not always correlate with the presence of disease. Anaplasma phagocytophilum DNA has been amplified from the blood of healthy cats for more than 10 weeks after experimental infection by exposure to Ixodes ticks.⁴¹

Cats can be infected by Rickettsia felis, Rickettsia conorii, and Rickettsia massiliae and have been shown to have antibodies against Rickettsia rickettsia.^42,43 Rickettsia felis is commonly amplified from fleas from cats and is associated with fever, headache, myalgia, and macular rash in humans.^44,45 However, further work is needed to determine whether spotted fever group Rickettsia spp. are associated with clinical illness in cats.

Blood culture, PCR assay on blood, and serologic testing can be used to assess individual cats for Bartonella spp. infection.⁴⁶ Cats that are culture-negative or PCR-negative and antibody-negative, and cats that are culture-negative or PCR-negative and antibody-positive, are probably not a source of flea, cat, or human infection. However, bacteremia can be intermittent, and false-negative culture or PCR results can occur, limiting the predictive value of a single battery of tests. Although serologic testing can be used to determine whether an individual cat has been exposed, both seropositive and seronegative cats can be bacteremic, limiting the diagnostic utility of serologic testing. Thus, testing healthy cats for Bartonella spp. infection is not currently recommended.^46,47 Testing should be reserved for cats with suspected clinical bartonellosis. Because Bartonella spp. infection is so common in healthy cats, even culture-positive or PCR-positive results do not prove clinical bartonellosis. For example, although we detected Bartonella spp. DNA in more cats with fever than in pair-matched cats without fever, the healthy cats were still commonly positive.⁴⁸ Combining serology with PCR in evaluation of cats with suspected bartonellosis is likely to give the best predictive value.

Cytauxzoon felis is usually easily identified on cytologic examination of blood smears or splenic aspirates during evaluation of clinically ill cats. Serologic testing is not commercially available currently. PCR can be used to amplify organism DNA from blood from cats that are cytologically negative.^49,50

Updated diagnostic testing recommendations for the two primary feline retroviruses, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV), were published in 2020.⁵¹ PCR can be used to confirm presence of FIV DNA in blood of cats and can be used to help differentiate seropositive FIV-vaccinated cats from cats with true infections. Most cats with clinical signs of FeLV infection are viremic, and so molecular diagnostic assays are not usually needed to diagnose this virus in clinical practice. However, some cats with regressive infection can be negative for FeLV antigen but positive for FeLV proviral DNA. Reactivation of progressive infection is possible in these cats and infection has been transmitted by blood transfusion.⁵² Amplification of FeLV proviral DNA from blood or bone marrow may be needed to prove associated diseases in some cats.

Wild and domestic cats are known to be infected by gammaherpesvirus 1.^53–55 Very little data is available concerning disease associations. However, in the United States, sick cats are more likely to be positive than healthy cats. The odds of being positive are higher in retrovirus-positive cats, male cats, and older cats.⁵³ Additional research will be needed to solidify disease associations with gammaherpesvirus 1 in cats.⁵⁶

Most RNA targets used in the RT-PCR assays that have been evaluated for the diagnosis of feline infectious peritonitis (FIP) have amplified both FIP virus and feline enteric coronavirus from the blood of some healthy cats. Thus, positive test results from blood do not always correlate with a diagnosis of FIP.⁵⁷ Performance of coronavirus PCR assays on fluids (effusion, aqueous humor, cerebrospinal fluid) or tissue aspirates is more likely to provide useful diagnostic information.⁵⁷

Toxoplasma gondii, Bartonella spp., FHV-1, and coronavirus are the organisms where DNA or RNA has been amplified most frequently from the aqueous humor of cats with endogenous uveitis.16,57,58 Since aqueous humor paracentesis could result in blood contamination, nucleic acids of agents present in the blood may be amplified. Although little is known about the predictive value of these assays when used with aqueous humor, the combination of molecular assays with local antibody production indices may aid in the diagnosis of some cases.

Molecular diagnostic assays have been used to aid in the diagnosis of Leptospira spp. infection in some cats.59,60 Leptospira spp. PCR assays are widely available in many countries and should be considered in the diagnosis of cats with acute renal injury in endemic areas. Molecular technology has also been used to investigate associations between chronic kidney disease and feline morbillivirus or feline foamy virus infections.^61,62 Molecular methods have been used to show that cats have a urinary system microbiome and that dysbiosis may be associated with chronic kidney disease.⁶³ How best to use this technology in clinical practice should be evaluated in future studies.