Evaluacion del PCR-Basado en los Test para Infecciones Patogenicas

8/16/2019 Evaluacion del PCR-Basado en los Test para Infecciones Patogenicas

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*A companion article titled “Poly-merase Chain Reaction Test Inter-pretation” appeared in the May 2008 issue.

he polymerase chain reaction (PCR)exponentially amplifies selected DNAsequences1 and has become an increas-

ingly popular tool for pathogen detection.2

PCR-based pathogen detection is rapid, candetect much smaller quantities of pathogenthan many other tests, is independent of hostresponse, can distinguish vaccination frompathogen infection, and is important in identi-fying pathogens such as viruses and rickettsiaethat are otherwise not easily isolated.3–5 Al-though PCR-based tests have great potential inpathogen detection, clinicians should considerthe limitations of PCR testing in general as

well as in working with particular pathogens.Correct and informed interpretation of testresults is critical to the successful use of PCR-based diagnostics.Clinicians should carefully ques-

tion the laboratory concerningstandard sample handling pro-cedures and testing protocols

because variations in DNA extraction protocols,PCR techniques,and reaction monitoring can alterthe validity of the test. False-negative and false-

positive test results can occur secondary to inhibi-tion of the reaction,6,7 sample handling errors, andthe limitations of laboratory techniques. In addi-tion, it is important to recognize that test resultsreflect only the presence or absence of pathogengenetic material in a sample. PCR pathogen testsrely on the detection of pathogen genetic material(DNA or RNA). A negative PCR test result willbe generated if no genetic material is present in thesample of an infected and clinically affected individual—for example, the apparent lack of Sarcocys-

tis neurona DNA in the cerebrospinal fluid (CSF)of horses with equine protozoal myeloencephalitis(EPM). 8 Conversely, the presence of genetic mate-rial does not guarantee the presence of live organ-isms; a horse with a positive PCR test result forSalmonella spp may not be actively shedding viableorganisms.9 Although PCR-based testing is apotentially powerful tool, because of its possiblelimitations and the misinterpretation of results, itshould be independently evaluated for eachpathogen of interest (Table 1).

COMPENDIUM EQUINE 308 July/August 2008

Evaluating PolymeraseChain Reaction–Based Testsfor Infectious Pathogens *

Julia Paxson,DVM, PhD Tufts University Cummings School of Veterinary Medicine

T

ABSTRACT: 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 aninappropriate 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.

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Table 1. PCR Testing Results for a Variety of Infectious Equine Pathogens

Pathogen Study PCR Test

PCR/Standard a Outcome

Conclusions+/+ +/- -/+ Total

Salmonella

spp

Cohen et al9

Amavisit et al14

Kurowski et al15

ssPCR(hisJ)

icPCR(ompC)rPCR(spaQ)

11

280

60

424

0

00

110

96230

PCR is sensitive in detecting this organism.

Monitor fecal inhibition using internal controls.DNA from nonviable organisms can result infalse-positive PCR results. Confirm results with culture.

Lawsonia spp Lavoie et al17 ssPCR 3 NA 2 29 PCR is prone to fecal inhibition. Use internalcontrols.

Clostridiumdifficile andClostridium perfringens

Magdesian et al26

Herholz et al22

ssPCR ssPCR

36NA

74NA

0NA

130NA

Sensitive for identification of pathogenicstrains.

Streptococcus equi

Newton et al38 nPCR 37 16 4 70 PCR is sensitive in detecting this organism.Identifies more animals that test positive thandoes culture.

Rhodococcus equi

Sellon et al34 NA 9 8 1 53 PCR is generally sensitive in detecting thisorganism.PCR tests distinguish pathogenic andnonpathogenic strains.

EHV1 Varrasso et al5 nPCR(glH) 20 0 1 71 PCR is sensitive in detecting this organism. Available as part of a respiratory panel.Best results are from nasopharyngeal swabs.

EHV4 Varrasso et al5

nPCR(glH) 4 1 0 7 Best results are from nasopharyngeal swabs.

Influenza A Quinlivan et al42 qRT-PCR(mat) 8 27 0 171 Best results are from nasopharyngeal swabs.

EIAV Nagarajan andSimard48

nPCR(gag) 81 7 0 122 PCR is sensitive in detecting this organismbut not yet accepted internationally.

West Nile virus

Johnson et al51 RT-nPCR(E) 10 3 0 73 PCR is unreliable in detecting this organism.Viremia does not correlate with clinical signs.

Potomachorse fever

Mott et al3 nPCR 17 0 3 27 Suggest conducting paired IFA titers andPCR testing.

Anaplasma spp Bullock et al54 nPCR(16s) 2 NA 2 375 PCR is sensitive in detecting this organismcompared with paired IFA titers.

Sarcocystis spp Miller andBernard8

ssPCR 9 3 116 259 PCR tests are unreliable in detecting thisorganism due to the lack of pathogen in theCSF.

a “Standard” indicates alternative diagnostic method (e.g., culture, viral isolation,parasite visualization, Coggins test for EIAV).b A sampling of commercial availability. The PCR methods used and the reported sensitivities and specificities may vary by laboratory: 1 =Colorado State University; 2 = University of Illinois; 3 = Iowa State University; 4 = Kansas State University; 5 = University of Minnesota; 6 =University of Missouri; 7 = Indiana Animal Disease Diagnostic Lab at Purdue University; 8 = University of Georgia; 9 = North Carolina StateUniversity; 10 = Dynagenics Veterinary Diagnostics; 11 = University of California, Davis; 12 = Cornell University; 13 = Equine Biodiagnostics,University of Kentucky.



SALMONELLA SPECIESSalmonella infection is a cause of potentially fatal entericdisease in horses and is associated with potentially highmortality rates of 42% to 44% related to virulentstrains.10,11 Active fecal shedding in the general horse

population is roughly 0.8%, as determined by singlefecal culture.12 However, fecal shedding within suscepti-ble populations, such as horses with gastrointestinal orrespiratory disease, appears to be increased.13 Monitor-ing sources of infection, especially asymptomatic carrieranimals that shed the organisms in their feces, is of par-ticular interest to large veterinary hospitals prone tonosocomial salmonellosis outbreaks.10 PCR testing hasbeen investigated as a method of identifying carrier ani-mals, following outbreaks, assessing sources of infection,and assessing the effectiveness of disinfection methods.

The reliability of fecal PCR-based Salmonella testinghas been addressed by targeting a variety of genes asso-ciated with pathogenic Salmonella spp and by a variety of DNA extraction techniques designed to reduce inhi-bition caused by urea and bilirubin compounds in feces.

The inhibition of PCR is monitored by including posi-tive internal controls. In one study, PCR targeting of theSalmonella ompC gene (encoding an outer membrane pro-tein) and construction of an appropriate targeted internalcontrol enabled direct monitoring of inhibition in eachPCR.14 In this clinical study, all samples that tested posi-

tive 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 negativeon culture.14 Real-time PCR has also been used toincrease the specificity of the reaction by including asequence-specific fluorogenic probe in addition to thestandard PCR primer pair. A study using real-time PCR to target the spaQ gene (a Salmonella pathogenicity genethat may be deleted in nonpathogenic environmental Sal-monella strains) demonstrated that PCR testing was moresensitive than culture for detecting pathogenic Salmonella

spp in a clinical setting.15

Results of multiple environmental sample testingstudies suggest that PCR test methods tend to be con-sistently more sensitive than culture for assessing possi-ble sources of Salmonella infection. This may beattributable to the fact that bacterial growth in culturecan be inhibited by the presence of disinfectants andthat PCR testing detects viable and nonviable organ-isms.16 Many teaching hospitals now routinely use PCR testing to detect possible environmental contamination, with the understanding that samples that test positive

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July/August 2008 COMPENDIUM EQUINE

Indications Availability b

Screening test for inpatients

Individual diagnosisEnvironmental testing

Yes—fecal

Laboratories: 1, 2, 4, 6,7,8,11

Individual diagnosis Alternative to culture of thisfastidious pathogen

Yes—fecalLaboratories: 3,6,7,11,12

Use for identification of pathogenic strains

Yes—fecalLaboratories: 1, 2, 3, 5, 7

Individual diagnosisScreening for inapparent carriers

Yes—nasal or TTALaboratories: 8, 11

Used for early individualdiagnosis Also useful for monitoring onendemic farms

Yes—TTA, nasalLaboratories: 8, 11

Useful for early diagnosis Yes—fetal tissue, nasalLaboratories: 1, 11, 12

Useful for early diagnosis Yes—nasalLaboratories: 1, 11Useful for early diagnosis Yes—nasal

Laboratories: 1,3,5,7,11Rapid screening method forcarrier animals

No

Postmortem only Not recommended as anantemortem diagnostic tool

Yes—brainLaboratories: 1,2,5,10,11

Useful for clinical diagnosis Yes—buffy

Laboratories: 10, 11Useful for clinical diagnosis Yes—buffy

Laboratories: 9, 10, 11Not recommended as anantemortem diagnostic tool atthis time

Yes—CSF Laboratories: 13

EHV = equine herpesvirus; EIAV = equine infectious anemia virus;icPCR = internal-control PCR; IFA = immunofluorescence assay;mPCR = multiplex PCR; NA = not available; nPCR = nested PCR;qRT-PCR = quantitative RT-PCR; rPCR = real-time PCR; RT-nPCR = reverse-transcription nested PCR; RT-PCR = reverse-transcriptionPCR; ssPCR = single-step PCR;TTA = transtracheal aspirate.



on PCR testing may reflect the presence of nonviableorganisms but that samples that test negative on PCR testing reflect a high probability that the environmenthas been effectively decontaminated.9

LAWSONIA INTRACELLULARIS Although bacterial culture has been the gold standardfor detecting Lawsonia intracellularis , culture of this fas-tidious intracellular bacterium is difficult and serialserologic testing is often used as an alternative diagnos-tic. Initial PCR testing of clinical samples was unreli-able because of the degree of inhibition caused by fecalby-products.17 In addition, serial PCR testing indicatesthat animals test negative within 4 days of initiatingantibiotic treatment but can be seropositive for up to 6months.18 Sample dilution, nested PCR, and the more

recent inclusion of internal mimics to detect inhibitionhave 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 andnested PCR testing, but the inclusion of internal mimics

to detect inhibition appears to have good sensitivity (24of 24 piglets that tested positive on histology also testedpositive on nested PCR testing).19,20

CLOSTRIDIUM DIFFICILE ANDCLOSTRIDIUM PERFRINGENSClostridial colitis can be associated with antibioticadministration in horses.21 PCR testing is used for rapididentification of toxigenic strains of Clostridium difficile and Clostridium perfringens.21,22 Enteropathogenicity of C. difficile strains in humans and horses has been associ-

ated with the presence of enterotoxin A (TcdA) andcytotoxin B (TcdB),23,24 and in horses, culture of C. diffi-cile does not correlate to the detection of pathogenictoxins.25 Toxigenic strains can be identified with com-mercial enzyme-linked fluorescent immunoassays fordetecting TcdA. However, the sensitivity of these assaysappears to be lower than the sensitivity of PCR assaysfor identifying the tcdA gene in clinical samples.26 Inaddition, recent reports of enteropathogenic strains thattest negative for TcdA and positive for TcdB in humanshave prompted the development of a real-time PCR test

for the tcdB gene, which has been shown to be presentin all human toxigenic strains of C. difficile.10,23 Usinginternal controls to monitor possible fecal inhibition,this real-time PCR test in humans reportedly has a sen-sitivity 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, andtypes A and C have been shown to produce β2 toxin(Cpb2). Because some strains of C. perfringens can becommensal in the equine large intestine, differentiationof pathogenic from nonpathogenic strains is crucial inmaking a diagnosis. Some studies have identified patho-genic C. perfringens through amplification of the entero-toxin gene.25 However, recent studies indicate that thepresence of β 2 toxin without the enterotoxin is sufficient

to cause gastrointestinal disease in horses.22,27

Further-more, studies indicate that exposure of some pathogenic

C. perfringens strains to the antibiotic gentamicin caninduce expression of the cpb2 gene, furthering the asso-ciation of C. perfringens overgrowth with antibiotic usein horses.28 These results indicate the need for PCR

testing of multiple possible toxin genes to most accu-rately identify possible pathogenic C. perfringens strains.

NEORICKETTSIA RISTICIIPotomac horse fever (equine monocytic ehrlichiosis) iscaused by the rickettsial bacterium Neorickettsia risticii and is associated with colitis in horses.29 The disease canbe diagnosed by a variety of methods, including bacter-ial culture, paired immunofluorescence assay (IFA)titers,30 and either peripheral blood mononuclear cell orfecal PCR testing.3 An IFA cannot distinguish between

past or present infection and vaccination and is associ-ated with a high rate of false-positive results.30 Com-pared with bacterial culture and IFA, the nested PCR test appears to detect the presence of the bacterium ear-lier in experimentally infected animals. However, whenthe nested PCR test was used in naturally infectedhorses, only 81% of animals that tested positive on cul-ture also tested positive on peripheral blood mononu-clear cell PCR testing, which may be attributable topoor recovery of Neorickettsia DNA from clinical speci-mens.3 More recently, a real-time PCR assay for blood



PCR-based diagnostic tests are useful for rapid and early detection of select equine pa



or fecal samples demonstrated good specificity as well assensitivity 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 ulcerativeenteritis in foals younger than 6 months and is tradition-ally diagnosed by culture and phenotypic analysis. How-ever, bacterial culture of transtracheal aspirate (TTA)samples can be inhibited by previous antimicrobial useand can be confounded by the presence of relatively non-pathogenic environmental R. equi strains.32 Compared with bacterial culture from TTA samples, PCR testingappears to be more sensitive.33,34 Creation of a multiplexassay to simultaneously target the Rhodococcus

spp–specific choE and vapA genes as well as a 16s riboso-mal internal control demonstrated high specificity andsensitivity, enabling differentiation of virulent and envi-ronmental strains as well as providing appropriate inter-nal controls.33 More recently, real-time PCR assays havebeen developed to target the vapA gene with even greatersensitivity than standard PCR testing, enabling detectionof much smaller quantities of the bacterium.35–37 Internal


amplification controls based on the choE gene have alsobeen included to monitor possible reaction inhibition.37

Real-time PCR studies have not yet documented successof these techniques in a clinical setting.

STREPTOCOCCUS EQUI SUBSP EQUIStreptococcus equi subsp equi is highly contagious andcommonly associated with barn-wide outbreaks of strangles that can be hard to control, with healthy carri-ers emerging in more than 50% of strangles outbreaks.38

Culture of either nasopharyngeal or guttural pouchswabs can be unrewarding during the early clinicalphase, and studies using nested PCR to target the S.equi subsp equi M protein gene indicate that PCR test-ing is more sensitive than culture.38 However, cliniciansmust recognize that PCR may be detecting amplifica-

tion of DNA from nonviable organisms and, therefore,the sample may not be from an infective source. Smallnumbers of false-negative PCR test results have beenattributed to inhibition secondary to excessive suppura-tive material in the samples.38 A multiplex PCR assay has also been established to rapidly differentiatebetween S. equi subsp zooepidemicus and S. equi subspequi isolates based on the newly described superanti-



July/August 2008


genic toxins SeeH and SeeI, which are present in S. equi subsp equi , but not in S. equi subsp zooepidemicus .39

EQUINE RESPIRATORY VIRUSESEquine influenza A2, equine herpesvirus (EHV) 1, and

EHV4 infections are considered to be some of the mostimportant contagious respiratory diseases in horses.40,41

Reverse-transcriptase PCR (RT-PCR) testing hasdemonstrated greater sensitivity than has virus isolationfrom nasal swab samples, which may be particularly important given the difficulties in isolating some fieldstrains of the influenza A virus sub-types.42 Multiplex PCR identifica-tion and differentiation of EHV1and EHV4 also appear to be sensi-tive.5 The development of a real-time

PCR-based test to quantify mRNAexpression (and thereby distinguishbetween latent and active infection)is promising.36 Because respiratory virus infections are common, work has been done to develop a compre-hensive 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-stepPCR, nested PCR, and RT-PCR tests.43 Most recently, a real-timePCR assay for EHV1 was developedand has shown good sensitivity andspecificity in a laboratory setting,although few data for positive clini-cal samples are available.44

EQUINE INFECTIOUSANEMIAVIRUS

Equine infectious anemia virus (EIAV)is an RNA virus of the family Retro- viridae. Animals infected with EIAV have an acute febrile response thatcan be fatal, followed by an inappar-ent carrier stage.45 During the asymp-tomatic phase, carrier animals havetraditionally been detected by use of an agar gel immunodeficiency assay,a Coggins test,46 or a competitiveELISA.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 detectmore EIAV-positive animals earlier in the course of infection and is as specific as the agar gel immunodefi-

ciency assay.48

EQUINE ENCEPHALITIS VIRUSESEastern, Western, and Venezuelan equine encephalitis viruses and West Nile virus are RNA viruses that causeinfectious encephalitis in horses.49 In patients infected





with these viruses, detection of the viral antigen or viralnucleic acid in serum is possible only if blood is collectedduring the viremic phase, which lasts 3 to 5 days andmay not correlate with the development of clinicalsigns.50 Exposure to the virus can be confirmed serologi-

cally using an ELISA. Definitive postmortem diagnosis via cell culture isolation from equine brain tissue can bedifficult,possibly because of low levels of viral replication within brain tissue.51 Several PCR tests, includingnested50–52 and real-time RT-PCR, 4 have been developedfor postmortem diagnosis using equine brain tissue. Bothnested and real-time RT-PCR have demonstrated a highsensitivity 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 antemortemdetection of West Nile virus in serum or CSF of clini-

cally ill animals. Viremia may occur before the develop-

ment 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 istraditionally made by visualization of morulae in granu-locytes or retrospectively based on an indirect IFA of paired serum samples. Studies using nested PCR ampli-fication of the 16s rRNA gene from peripheral bloodmononuclear cells suggest that PCR is a sensitivemethod of detection that can detect the presence of theorganism several days before morulae can be identified

with light microscopy.54,55

SARCOCYSTIS NEURONA The neurologic disease EPM can develop when S. neu-rona protozoa invade the central nervous system. A diag-nosis is traditionally based on a CSF immunoblotbecause it is believed that the presence of host antibodiesin the CSF (in an uncontaminated sample with minimalblood–brain barrier damage) is most closely correlated with clinical disease.24 A PCR test to detect S. neurona inCSF has been developed in an attempt to overcome lim-

itations of the immunoblot tests. The test appears to beof little use in antemortem diagnosis because it has ahigh rate of negative results in possibly infected animals(e.g., one study demonstrated 116 results of CSF thattested positive on Western blot testing but negative on

PCR testing).8 S. neurona merozoites are usually foundintracellularly or within the parenchyma of the spinalcord and brain, and parasite antigen and DNA are rarely found in CSF.8 However, PCR testing has been found tobe useful in postmortem identification of parasites fromneuronal tissue.36 In the absence of parasite DNA, false-negative PCR test results are likely. Unlike immunoblottests that yield a dichotomous result (positive or nega-tive), a recently developed indirect fluorescent antibody test enables more precise quantification of both serumand CSF antibody levels. A recent study demonstrated

correlation between serum and CSF indirect fluorescent

antibody test antibody detection, and the authors suggestthat, in most cases, CSF or serum antibody quantifica-tion may be sufficient for determining the probability of

infection.56

CONCLUSIONPCR-based diagnostic tests have become increasingly popular in equine medicine for the rapid and accuratedetection of infectious pathogens. PCR-based tests canbe extremely sensitive diagnostic tools, allowing patho-gen detection earlier in the course of the disease (e.g.,infection with A. phagocytophila 54 or EIAV 48). However,because fragmented nucleic acids from nonviable cellsor viruses can be readily amplified using PCR, PCR-

based tests cannot identify animals that harbor nonvi-able pathogen and are no longer contagious (e.g., horses with Salmonella infection44). False-negative PCR testresults f rom infected animals can occur when the testedsample 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 ani-mals that have passed the short-lived viremic phase,50,51

lack of the targeted virulence genes in some pathogenicstrains of R. equi 34). Correct interpretation of PCR testresults and a thorough knowledge of the protocol used

The validity of each PCR-based diagnostic test depends on the application,the PCR protocol that is used, and the correct interpretation of results.



July/August 2008 COMPENDIUM EQUINE


are critical in evaluating PCR-based tests. False-positivePCR test results from uninfected animals are possible if contaminating DNA is amplified; therefore, PCR testresults should be monitored by including controls, andfalse-positive results should be limited by careful sample

collection and good laboratory practices. False-negativePCR results from infected animals can result from low DNA copy number, poor primer performance and thepresence of inhibitory substances in the sample (e.g.,urea, heme), and prior antimicrobial use. Better DNAextraction techniques, the inclusion of appropriate con-trols, and techniques such as nested PCR have beenused to monitor PCR test success and reduce potentialproblems.3

PCR-based diagnostic tests appear to be useful inrapidly detecting Salmonella spp ,14 and new develop-

ments in PCR testing for other fecal pathogens (e.g.,Lawsonia and Clostridium spp) have increased the valueof these tests.26 PCR-based tests are now routinely usedfor rapid and early detection of S. equi subsp equi,38 R.equi,33 N. risticii,3 A. phagocytophila,54 and equine respira-tory viruses.42 Although international regulations stillstipulate use of the traditional agar gel immunodefi-ciency assay or Coggins test, PCR-based tests for EIAV are also promising.48 PCR-based antemortem diagnosisof EPM8 and viral encephalitis51 from CSF does notappear to be reliable.

Because of the variety of factors that can influence the validity of PCR results, it is important to understand thepossible limitations of the test in general and in regardto the particular pathogen of interest. Table 1 summa-rizes the use of PCR tests in detecting a variety of infec-tious equine pathogens. When used appropriately, themany variations on the basic PCR protocol can greatly enhance the clinical usefulness of the particular test.Because different PCR tests are available, practitionersshould discuss with their laboratory which protocol touse for the pathogen of interest and how to best inter-

pret the results from a given PCR test.ACKNOWLEDGMENTS The author thanks Drs. Melissa Mazan, Mary Rose Paradis, Lois Wetmore,Daniela Bedenice, and Rose Nolen-Walston for their comments.

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31. Pusterla N, Leutenegger CM, Sigrist B, et al. Detection and quantitation of Ehrlichia ristici i genomic DNA in infected horses and snails by real-timePCR.Vet Parasitol 2000;90(1-2):129-135.

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1. Which statement regarding PCR is incorrect?a. PCR is the most sensitive diagnostic tool for all

equine infectious pathogens.b. False-negative and false-positive PCR test results can

occur.c. Pathogen genetic material (DNA or RNA) must be

present in the tested sample.d. Positive PCR test results can occur in animals with-

out active infection.

2. PCR testing is not a valid diagnostic modality for

a. confirming active infection.b. screening horse populations for asymptomatic car-rier animals that shed a pathogen in their feces.

c. detecting infection of fastidious pathogens that arehard to culture or otherwise isolate.

d. confirming infection with a virulent (rather than envi-ronmental) strain of bacteria.

3. To confirm active Salmonella infection in a horse with a positive PCR test result, a clinician shoulda. obtain a positive culture result.b. obtain positive antibody titer results.

ARTICLE #1 CE TEST This article qualifies for 2 contact hours of continuing education credit from the Auburn University College of Veterinary Medicine.Subscribers may take individualCE tests or sign up for our annual CE program.Those who wish to apply this credit to fulfill state relicensurerequirements should consult their respective stateauthorities regarding the applicability of this program.CE subscribers can take CE tests online and get real-timescores at CompendiumEquine.com .

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c. confirm the presence of associated clinical signs.d. confirm the presence of leukopenia.

4. PCR inhibition caused by fecal samplesa. can be monitored with internal controls.

b. is rare.c. can be associated with prior antimicrobial use.d. is more common in animals with diarrhea.

5. Use of PCR to monitor environmental sources of Salmonella sppa. is not inhibited by the prior use of detergents.b. may reflect the presence of nonviable organisms.c. is more sensitive than culture.d. all of the above

6. Use of PCR to detect R. equia. is straightforward because all Rhodococcus strains are equally pathogenic.

b. is less sensitive than bacterial culture of TTA fluid.c. is rarely diagnostic because genetic material is rarely present in TTA or

fecal samples.d. may have increased diagnostic value if the pathogenic vapA gene is

targeted.

7. Use of PCR to detect S. equi subsp equia. can be inhibited by prior use of antimicrobials.b. always indicates the presence of active infection.c. is more sensitive than bacterial culture.d. is not subject to inhibition in purulent samples.

8. Which statement regarding viral disease is incorrect?a. For some viral diseases, rising paired IFA titers with a negative PCR test

result may still indicate active infection.b. For some respiratory viral diseases, PCR testing may be more sensitive

for testing nasal swabs than blood.c. For viral encephalitis diseases, PCR testing is more sensitive for testing

blood than brain tissue.d. Both RNA and DNA viruses can be detected using PCR techniques.

9. PCR-based antemortem diagnosis of encephalitis virus infection ishampered bya. inhibition of PCR.b. a short viremic phase.c. large numbers of false-positive PCR results.d. overwhelming inflammation.

10. In diagnosing diseases such as EPM, PCR testing can be ineffectivebecausea. PCR is generally inhibited in clinical samples.b. genetic material is not usually present in the samples.c. clinical signs of disease precede the ability of PCR to detect the

pathogen.d. it is effective for testing CSF samples, but not blood samples.



Evaluacion del PCR-Basado en los Test para Infecciones Patogenicas

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