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Equine July/August 2008 (Vol 3, No 6)

Evaluating Polymerase Chain Reaction-Based Tests for Infectious Pathogens

by Julia Paxson

    CETEST This course is approved for 2.0 CE credits

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    A companion article titled "Polymerase Chain Reaction Test Interpretation" appeared in the May 2008 issue.


    Polymerase chain reaction (PCR)—based diagnostic tests can allow rapid and sensitive detection of equine infectious pathogens. However, PCR is not without limitations and is an inappropriate diagnostic tool for some diseases. This review analyzes the advantages and limitations of PCR-based diagnostic tests for several important equine infectious pathogens and suggests questions for practitioners to consider when choosing a commercial PCR test for clinical diagnosis of a particular infectious pathogen.

    The polymerase chain reaction (PCR) exponentially amplifies selected DNA sequences1 and has become an increasingly popular tool for pathogen detection.2 PCR-based pathogen detection is rapid, can detect much smaller quantities of pathogen than many other tests, is independent of host response, can distinguish vaccination from pathogen infection, and is important in identifying pathogens such as viruses and rickettsiae that are otherwise not easily isolated.3-5 Al­though PCR-based tests have great potential in pathogen detection, clinicians should consider the limitations of PCR testing in general as well as in working with particular pathogens.

    Correct and informed interpretation of test results is critical to the successful use of PCR-based diagnostics. Clinicians should carefully question the laboratory concerning standard sample handling procedures and testing protocols because variations in DNA extraction protocols, PCR techniques, and reaction monitoring can alter the validity of the test. False-negative and false-positive test results can occur secondary to inhibition of the reaction,6,7 sample handling errors, and the limitations of laboratory techniques. In addition, it is important to recognize that test results reflect only the presence or absence of pathogen genetic material in a sample. PCR pathogen tests rely on the detection of pathogen genetic material (DNA or RNA). A negative PCR test result will be generated if no genetic material is present in the sample of an infected and clinically affected individual—for example, the apparent lack of Sarcocystis neurona DNA in the cerebrospinal fluid (CSF) of horses with equine protozoal myeloencephalitis (EPM).8 Conversely, the presence of genetic material does not guarantee the presence of live organisms; a horse with a positive PCR test result for Salmonella spp may not be actively shedding viable organisms.9 Although PCR-based testing is a potentially powerful tool, because of its possible limitations and the misinterpretation of results, it should be independently evaluated for each pathogen of interest (Table 1).

    Salmonella Species

    Salmonella infection is a cause of potentially fatal enteric disease in horses and is associated with potentially high mortality rates of 42% to 44% related to virulent strains.10,11 Active fecal shedding in the general horse population is roughly 0.8%, as determined by single fecal culture.12 However, fecal shedding within susceptible populations, such as horses with gastrointestinal or respiratory disease, appears to be increased.13 Monitoring sources of infection, especially asymptomatic carrier animals that shed the organisms in their feces, is of particular interest to large veterinary hospitals prone to nosocomial salmonellosis outbreaks.10 PCR testing has been investigated as a method of identifying carrier animals, following outbreaks, assessing sources of infection, and assessing the effectiveness of disinfection methods. The reliability of fecal PCR-based Salmonella testing has been addressed by targeting a variety of genes associated with pathogenic Salmonella spp and by a variety of DNA extraction techniques designed to reduce inhibition caused by urea and bilirubin compounds in feces.

    The inhibition of PCR is monitored by including positive internal controls. In one study, PCR targeting of the Salmonella ompC gene (encoding an outer membrane protein) and construction of an appropriate targeted internal control enabled direct monitoring of inhibition in each PCR.14 In this clinical study, all samples that tested positive on culture also tested positive on a single-step PCR test. In addition, the authors noted a large number of samples that tested positive on PCR testing and negative on culture.14 Real-time PCR has also been used to increase the specificity of the reaction by including a sequence-specific fluorogenic probe in addition to the standard PCR primer pair. A study using real-time PCR to target the spaQ gene (a Salmonella pathogenicity gene that may be deleted in nonpathogenic environmental Salmonella strains) demonstrated that PCR testing was more sensitive than culture for detecting pathogenic Salmonella spp in a clinical setting.15

    Results of multiple environmental sample testing studies suggest that PCR test methods tend to be consistently more sensitive than culture for assessing possible sources of Salmonella infection. This may be attributable to the fact that bacterial growth in culture can be inhibited by the presence of disinfectants and that PCR testing detects viable and nonviable organisms.16 Many teaching hospitals now routinely use PCR testing to detect possible environmental contamination, with the understanding that samples that test positive on PCR testing may reflect the presence of nonviable organisms but that samples that test negative on PCR testing reflect a high probability that the environment has been effectively decontaminated.9

    Lawsonia intracellularis

    Although bacterial culture has been the gold standard for detecting Lawsonia intracellularis, culture of this fastidious intracellular bacterium is difficult and serial serologic testing is often used as an alternative diagnostic. Initial PCR testing of clinical samples was unreliable because of the degree of inhibition caused by fecal by-products.17 In addition, serial PCR testing indicates that animals test negative within 4 days of initiating antibiotic treatment but can be seropositive for up to 6 months.18 Sample dilution, nested PCR, and the more recent inclusion of internal mimics to detect inhibition have led to greater success in detecting L. intracellularis in piglets. In these studies, inhibition (as assessed by failure to amplify the mimic) was observed in roughly 10% of cases, even with the use of diluted samples and nested PCR testing, but the inclusion of internal mimics to detect inhibition appears to have good sensitivity (24 of 24 piglets that tested positive on histology also tested positive on nested PCR testing).19,20

    Clostridium difficile and Clostridium perfringens

    Clostridial colitis can be associated with antibiotic administration in horses.21 PCR testing is used for rapid identification of toxigenic strains of Clostridium difficile and Clostridium perfringens.21,22 Enteropathogenicity of C. difficile strains in humans and horses has been associated with the presence of enterotoxin A (TcdA) and cytotoxin B (TcdB),23,24 and in horses, culture of C. difficile does not correlate to the detection of pathogenic toxins.25 Toxigenic strains can be identified with commercial enzyme-linked fluorescent immunoassays for detecting TcdA. However, the sensitivity of these assays appears to be lower than the sensitivity of PCR assays for identifying the tcdA gene in clinical samples.26 In addition, recent reports of enteropathogenic strains that test negative for TcdA and positive for TcdB in humans have prompted the development of a real-time PCR test for the tcdB gene, which has been shown to be present in all human toxigenic strains of C. difficile.10,23 Using internal controls to monitor possible fecal inhibition, this real-time PCR test in humans reportedly has a sensitivity of 100% and a specificity of 94%.23

    C. perfringens isolates are commonly classified as types A through E based on toxin production. In addition, type A isolates produce C. perfringens enterotoxin, and types A and C have been shown to produce b2 toxin (Cpb2). Because some strains of C. perfringens can be commensal in the equine large intestine, differentiation of pathogenic from nonpathogenic strains is crucial in making a diagnosis. Some studies have identified pathogenic C. perfringens through amplification of the enterotoxin gene.25 However, recent studies indicate that the presence of b2 toxin without the enterotoxin is sufficient to cause gastrointestinal disease in horses.22,27 Furthermore, studies indicate that exposure of some pathogenic C. perfringens strains to the antibiotic gentamicin can induce expression of the cpb2 gene, furthering the association of C. perfringens overgrowth with antibiotic use in horses.28 These results indicate the need for PCR testing of multiple possible toxin genes to most accurately identify possible pathogenic C. perfringens strains.

    Neorickettsia risticii

    Potomac horse fever (equine monocytic ehrlichiosis) is caused by the rickettsial bacterium Neorickettsia risticii and is associated with colitis in horses.29 The disease can be diagnosed by a variety of methods, including bacterial culture, paired immunofluorescence assay (IFA) titers,30 and either peripheral blood mononuclear cell or fecal PCR testing.3 An IFA cannot distinguish between past or present infection and vaccination and is associated with a high rate of false-positive results.30 Compared with bacterial culture and IFA, the nested PCR test appears to detect the presence of the bacterium earlier in experimentally infected animals. However, when the nested PCR test was used in naturally infected horses, only 81% of animals that tested positive on culture also tested positive on peripheral blood mononuclear cell PCR testing, which may be attributable to poor recovery of Neorickettsia DNA from clinical specimens.3 More recently, a real-time PCR assay for blood or fecal samples demonstrated good specificity as well as sensitivity comparable with that of nested PCR, but it was not as sensitive as culture.31

    Rhodococcus equi

    Rhodococcus equi is associated with the development of severe pyogranulomatous pneumonia and ulcerative enteritis in foals younger than 6 months and is traditionally diagnosed by culture and phenotypic analysis. However, bacterial culture of transtracheal aspirate (TTA) samples can be inhibited by previous antimicrobial use and can be confounded by the presence of relatively nonpathogenic environmental R. equi strains.32 Compared with bacterial culture from TTA samples, PCR testing appears to be more sensitive.33,34 Creation of a multiplex assay to simultaneously target the Rhodococcus spp-specific choE and vapA genes as well as a 16s ribosomal internal control demonstrated high specificity and sensitivity, enabling differentiation of virulent and environmental strains as well as providing appropriate internal controls.33 More recently, real-time PCR assays have been developed to target the vapA gene with even greater sensitivity than standard PCR testing, enabling detection of much smaller quantities of the bacterium.35-37 Internal amplification controls based on the choE gene have also been included to monitor possible reaction inhibition.37 Real-time PCR studies have not yet documented success of these techniques in a clinical setting.

    Streptococcus equi Subsp equi

    Streptococcus equi subsp equi is highly contagious and commonly associated with barn-wide outbreaks of strangles that can be hard to control, with healthy carriers emerging in more than 50% of strangles outbreaks.38 Culture of either nasopharyngeal or guttural pouch swabs can be unrewarding during the early clinical phase, and studies using nested PCR to target the S. equi subsp equi M protein gene indicate that PCR testing is more sensitive than culture.38 However, clinicians must recognize that PCR may be detecting amplification of DNA from nonviable organisms and, therefore, the sample may not be from an infective source. Small numbers of false-negative PCR test results have been attributed to inhibition secondary to excessive suppurative material in the samples.38 A multiplex PCR assay has also been established to rapidly differentiate between S. equi subsp zooepidemicus and S. equi subsp equi isolates based on the newly described superantigenic toxins SeeH and SeeI, which are present in S. equi subsp equi, but not in S. equi subsp zooepidemicus.39

    Equine Respiratory Viruses

    Equine influenza A2, equine herpesvirus (EHV) 1, and EHV4 infections are considered to be some of the most important contagious respiratory diseases in horses.40,41 Reverse-transcriptase PCR (RT-PCR) testing has demonstrated greater sensitivity than has virus isolation from nasal swab samples, which may be particularly important given the difficulties in isolating some field strains of the influenza A virus subtypes.42 Multiplex PCR identification and differentiation of EHV1 and EHV4 also appear to be sensitive.5 The development of a real-time PCR-based test to quantify mRNA expression (and thereby distinguish between latent and active infection) is promising.36 Because respiratory virus infections are common, work has been done to develop a comprehensive panel of commercial PCR tests that can detect EHV1, EHV2, EHV4, EHV5, equine adenovirus 1, equine adenovirus 2, equine arteritis virus, and equine rhinitis A virus by using a combination of single-step PCR, nested PCR, and RT-PCR tests.43 Most recently, a real-time PCR assay for EHV1 was developed and has shown good sensitivity and specificity in a laboratory setting, although few data for positive clinical samples are available.44

    Equine Infectious Anemia Virus

    Equine infectious anemia virus (EIAV) is an RNA virus of the family Retroviridae. Animals infected with EIAV have an acute febrile response that can be fatal, followed by an inapparent carrier stage.45 During the asymp­tomatic phase, carrier animals have traditionally been detected by use of an agar gel immunodeficiency assay, a Coggins test,46 or a competitive ELISA.47 The principal drawback of these diagnostic tests is their reliance on the presence of specific antibodies that can be absent in early infection. Nested PCR testing for proviral DNA appears to detect more EIAV-positive animals earlier in the course of infection and is as specific as the agar gel immunodeficiency assay.48

    Equine Encephalitis Viruses

    Eastern, Western, and Venezuelan equine encephalitis viruses and West Nile virus are RNA viruses that cause infectious encephalitis in horses.49 In patients infected with these viruses, detection of the viral antigen or viral nucleic acid in serum is possible only if blood is collected during the viremic phase, which lasts 3 to 5 days and may not correlate with the development of clinical signs.50 Exposure to the virus can be confirmed serologically using an ELISA. Definitive postmortem diagnosis via cell culture isolation from equine brain tissue can be difficult, possibly because of low levels of viral replication within brain tissue.51 Several PCR tests, including nested50-52 and real-time RT-PCR,4 have been developed for postmortem diagnosis using equine brain tissue. Both nested and real-time RT-PCR have demonstrated a high sensitivity in detecting the virus in brain tissue compared with the sensitivity of culture.4 However, the same PCR techniques are not reliable when used for antemortem detection of West Nile virus in serum or CSF of clinically ill animals. Viremia may occur before the development of clinical signs,51 and serology is currently the only clinically useful method of antemortem diagnosis of West Nile encephalitis in horses.

    Anaplasma phagocytophilum

    Anaplasma phagocytophilum (formerly Ehrlichia equi) causes tick-borne vasculitis in horses.53 A diagnosis is traditionally made by visualization of morulae in granulocytes or retrospectively based on an indirect IFA of paired serum samples. Studies using nested PCR amplification of the 16s rRNA gene from peripheral blood mononuclear cells suggest that PCR is a sensitive method of detection that can detect the presence of the organism several days before morulae can be identified with light microscopy.54,55

    Sarcocystis neurona

    The neurologic disease EPM can develop when S. neurona protozoa invade the central nervous system. A diagnosis is traditionally based on a CSF immunoblot because it is believed that the presence of host antibodies in the CSF (in an uncontaminated sample with minimal blood-brain barrier damage) is most closely correlated with clinical disease.24 A PCR test to detect S. neurona in CSF has been developed in an attempt to overcome limitations of the immunoblot tests. The test appears to be of little use in antemortem diagnosis because it has a high rate of negative results in possibly infected animals (e.g., one study demonstrated 116 results of CSF that tested positive on Western blot testing but negative on PCR testing).8 S. neurona merozoites are usually found intracellularly or within the parenchyma of the spinal cord and brain, and parasite antigen and DNA are rarely found in CSF.8 However, PCR testing has been found to be useful in postmortem identification of parasites from neuronal tissue.36 In the absence of parasite DNA, false-negative PCR test results are likely. Unlike immunoblot tests that yield a dichotomous result (positive or negative), a recently developed indirect fluorescent antibody test enables more precise quantification of both serum and CSF antibody levels. A recent study demonstrated correlation between serum and CSF indirect fluorescent antibody test antibody detection, and the authors suggest that, in most cases, CSF or serum antibody quantification may be sufficient for determining the probability of infection.56


    PCR-based diagnostic tests have become increasingly popular in equine medicine for the rapid and accurate detection of infectious pathogens. PCR-based tests can be extremely sensitive diagnostic tools, allowing patho­gen detection earlier in the course of the disease (e.g., infection with A. phagocytophila54 or EIAV48). However, because fragmented nucleic acids from nonviable cells or viruses can be readily amplified using PCR, PCR-based tests cannot identify animals that harbor nonviable pathogen and are no longer contagious (e.g., horses with Salmonella infection44). False-negative PCR test results from infected animals can occur when the tested sample does not contain pathogen genetic material (e.g., lack of S. neurona schizonts in CSF samples,24 lack of encephalitis virus particles in the serum of infected animals that have passed the short-lived viremic phase,50,51 lack of the targeted virulence genes in some pathogenic strains of R. equi34). Correct interpretation of PCR test results and a thorough knowledge of the protocol used are critical in evaluating PCR-based tests. False-positive PCR test results from uninfected animals are possible if contaminating DNA is amplified; therefore, PCR test results should be monitored by including controls, and false-positive results should be limited by careful sample collection and good laboratory practices. False-negative PCR results from infected animals can result from low DNA copy number, poor primer performance and the presence of inhibitory substances in the sample (e.g., urea, heme), and prior antimicrobial use. Better DNA extraction techniques, the inclusion of appropriate controls, and techniques such as nested PCR have been used to monitor PCR test success and reduce potential problems.3

    PCR-based diagnostic tests appear to be useful in rapidly detecting Salmonella spp,14 and new developments in PCR testing for other fecal pathogens (e.g., Lawsonia and Clostridium spp) have increased the value of these tests.26 PCR-based tests are now routinely used for rapid and early detection of S. equi subsp equi,38 R. equi,33 N. risticii,3 A. phagocytophila,54 and equine respiratory viruses.42 Although international regulations still stipulate use of the traditional agar gel immunodeficiency assay or Coggins test, PCR-based tests for EIAV are also promising.48 PCR-based antemortem diagnosis of EPM8 and viral encephalitis51 from CSF does not appear to be reliable.

    Because of the variety of factors that can influence the validity of PCR results, it is important to understand the possible limitations of the test in general and in regard to the particular pathogen of interest.  Table 1 summarizes the use of PCR tests in detecting a variety of infectious equine pathogens. When used appropriately, the many variations on the basic PCR protocol can greatly enhance the clinical usefulness of the particular test. Because different PCR tests are available, practitioners should discuss with their laboratory which protocol to use for the pathogen of interest and how to best interpret the results from a given PCR test.


    The author thanks Drs. Melissa Mazan, Mary Rose Paradis, Lois Wetmore, Daniela Bedenice, and Rose Nolen-Walston for their comments.

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