Rapid detection of toxigenic Clostridium difficile in fecal samples by magnetic immuno PCR assay

0095-1137/94/$04.00+0

Copyright ©) 1994,AmericanSocietyforMicrobiology

Rapid Detection of Toxigenic

Clostridium

difficile

in

Fecal

Samples by Magnetic

Immuno

PCR Assay

MAURICEJ. H. M. WOLFHAGEN,l 2* ADC. FLUIT,1'3 RUURD TORENSMA,' 3 MIRIAMJ. J. G.POPPELIER,"3 ANDJAN VERHOEF'

Eijkman-Winkler

Laboratoryfor Medical Microbiology, University Hospital Utrecht, 3508 GA Utrecht,1 Hanzelaboratory for Medical Microbiology and Infectious Diseases, 8021 AM Zwolle,2 and U-Gene Research by,

3584 CJ

Utrecht,3

The Netherlands

Received 17 November1993/Returned for modification7February 1994/Accepted 5April 1994

Rapid detection oftoxigenic Clostridium

dificile

in fecal samples was accomplished with the magnetic immuno PCR assay (MIPA). Elaborate DNAextraction techniques were unnecessary. First,wegenerated a mouse monoclonal antibody (MAb) reactive with only C.

difficile,

Clostridium sordellii, and Clostridium bifermentans. Then, magnetic beads were coatedwith the MAb,incubatedwith fecal samplestoallowbinding

withC.

difficile,

extracted from the stool with a magnet, and processedinthePCRwithprimersspecific forthe

toxin Bgene. AfteroptimizingMIPAbyraisingthe number ofPCRcyclesfrom35to40 andadding Chelex100 to thePCRmixture, wefound asensitivity of 96.7%,aspecificityof100%,apositive predictivevalue of100%, and a negative predictive value of 94.1%whencompared with the culture ofcytotoxic C.

difficile

from fecal samples. MIPA is a rapid, easy, and sensitive PCR method fordemonstrating thepresence oftoxigenic C. difficile in stool samples andavoids thedisadvantage of elaborateextraction of DNA fromfecal samples.

Toxigenic Clostridium difficile can be the cause of various

clinical syndromes, ranging from asymptomatic colonization via mild antibiotic-associated diarrhea topseudomembranous colitis,whichcanprogresstotoxic megacolonandsubsequent death (3). Although diseases caused by this organism have been known since 1974 (25), the diagnosis of the cause of

antibiotic-associated diarrhea still poses manyproblems. The toxin Bassayof stoolsamples bythe tissue culturecytotoxicity

assay is still considered to be the "gold standard" (3), even

thoughPetersonetal.(23)foundasensitivityofonly67% and aspecificity of 99% for the demonstrationoftoxinB in stool

samples, while they found a sensitivity of culture of stool

samples for C. difficile of97% anda specificity of93% (23).

Furthermore, pseudomembranous colitiscanbe diagnosed by

proctosigmoidoscopy inpatientswithout apositivestool cyto-toxicityassayresult (13).

In contrast to the tissue culture cytotoxicity assay,

demon-stration of thepresenceof C.difficileinstool culturescaneasily

be performed by using commercially available culture media based on the formula described by George et al. (11).

How-ever,bothnontoxigenic andtoxigenic C. difficilestrainscanbe

cultured in this medium. This means that for a definite diagnosis,thecapacitiesof theseisolatestoproduce toxinmust

still be determined. This can be done not only by the tissue culturecytotoxicity assaybut also by demonstrating the pres-enceofthe genesencoding eithertoxinAortoxin Borboth.

We and several others have found that the presence of the

genes encoding for toxin A and toxin B correlates with the

presenceoftoxins,withveryfewexceptions (10,17,22, 31-34).

Two methods for the direct identification of the genes

encodingfor toxins have been described: colony blot hybrid-ization, which is performed on replica primary plates

inocu-lated withstool samples (29),and the detection of toxigenicC. difficile in stool samples bythe PCR (15, 18). Both methods

*Corresponding author. Mailing address: Eijkman-Winkler

Labo-ratory for Medical Microbiology, University Hospital Utrecht, P.O. Box85500,roomG.04.5 15,Heidelberglaan 100, 3508 GA Utrecht, The Netherlands. Phone: + -31-30507630. Fax: + -31-30541770.

have drawbacks. Colony blot hybridization requires that the agarplate be incubated for24h, while the PCR itself is simple, but the samplepreparationis laborious (4, 14, 15, 18).

Recently, a magnetic immuno PCR assay (MIPA) was described. The MIPAallowed for the detection of Salmonella spp. in fecalsamples(28)and avoided the needto extractDNA from the stool sample. This method uses magnetic beads coated with antibodiesto extractthe targetorganismfrom the

I sample. In this way, the advantages ofPCRcan be exploited

without the disadvantageof elaborate sample preparation. To testthe applicability of this methodology for the detection of C. difficile, we first produced a mouse monoclonal antibody

(MAb) reactive with all known C.

difficile

serotypes(7, 8) and then usedMIPA todemonstratetoxigenicC.difficile directlyin fecalsamples.

MATERIALSAND METHODS

Bacterialstrains.TheC. difficilereferencestrains, including the 10 serotypes thatare nowknown,werepurchasedfrom the American Type Culture Collection (ATCC 9689, ATCC 43593, ATCC 43594, and ATCC 43596toATYC43603)(7,8). Theotherstrains and clinical isolateswere kindly donated by various Dutch hospitals and the Dutch National Institute for Public Health and Environmental Protection (Rijksinstituut voorVolkgezondheidenMilieuhygiene),Bilthoven, The Neth-erlands.

Clinical specimens were frozen immediately upon receipt. Toisolate strains from clinical specimens, fecal samples were platedontoC.

difficile

agar (Oxoid, Basingstoke, United King-dom) containing500 mgofcycloserineper literand 16 mgof cefoxitin (Oxoid) per liter, and the plates were incubated anaerobically for 48 h at 37°C. Cultures suspect for the presence ofC. difficilewere identified asdescribed by Holde-man etal. (16).

Tissue culturecytotoxicity assay. The tissue culture cytotox-icity assaywasperformed on all fecal specimensas described by Allen(1).Briefly,cytotoxicitywasdeterminedonVerocells grown in 96-well plates. The assay was considered positive

1629

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when cells showed at least a 50% cytopathic effect and could be neutralized byClostridiumsordellii antitoxin (Wellcome, Tem-ple Hill, United Kingdom).

Generation of MAbs. Female BALB/c mice (ages, 6 to 8 weeks) were used for the generation of MAbs. These mice wereimmunized weekly, first subcutaneously with the different live C. difficile strains from ATCC and later intraperitoneally with only toxin-negative strains. A total of 10 CFU per immunization was used. Spleen cells obtained from immunized mice were collected and fused with the Sp2/0 myeloma cell line. Supernatants from wells containing growing hybridomas were screened foragglutinating antibodies by slide agglutina-tion, with live ATCC strains used as the antigen.

Immunoblots.Thereactivities of thestrains in immunoblots weretested asdescribed by McKay et al. (21). Briefly, a loopful ofC. difficile ATCC 9689, cultured overnight on Reinforced

Clostridial Agar (Oxoid), was boiled in sample buffer (4%

sodium dodecyl sulfate [SDS], 5% dithiothreitol, and 40% glycerol in 0.1 MTris-HCl [pH 7.0]) for 5 min. SDS-polyacryl-amide gel electrophoresis was performed as described by Laemmli (19). Proteins were analyzed after blotting onto nitrocellulose filters(26).Filters were blocked with3% gelatin and 0.05% Tween 20 in phosphate-buffered saline (pH 7.4) and wereprobed with the supernatant of hybridoma-contain-ing wells. After washhybridoma-contain-ing, a goat anti-mouse immunoglobulin G

(IgG)labeledwith horseradish peroxidase(Southern Biotech-nology Associates, Birmingham,Ala.)wasadded and thefilters wereincubated for 1 h at 37°C. Final results were obtained by incubating the filters in3,3-diaminobenzidine tetrahydrochlo-ride (0.5 mg/ml)-0.075% H202.

Magneticimmunoseparation.MagnisortMmagnetic chro-mium dioxide particles coated with goat immunoglobulins specific for murine immunoglobulin (IgG and IgM; Dupont,

Wilmington, Del.) were incubated with 75 ,ul of hybridoma culture supernatant at room temperature for 15 min with continuousshaking.Themagneticparticleswererecovered by magnetic force,and the supernatantwasdiscarded.

Stoolspecimenswerediluted 1:20 with saline andvigorously

shakentoobtainahomogenized sample.After thesamplewas allowedtosettle, 100

,ll

ofsupernatantwasincubated with the

magnetic particles for 15 min at room temperature with continuousshaking.Theparticleswere extractedby magnetic force,washed threetimeswithsaline,andthenresuspendedin 100 ,ul ofdistilledwaterwith orwithout 5% (wt/vol) Chelex 100(Bio-Rad,Richmond,Calif.) (28).Thiswasthenprocessed

forPCR.

PCR. The PCRwasperformedasdescribedbyFluitetal.(9, 10). Inshort, theresuspendedbeadswereheated for 5 min at 95°C to lyse the bound bacteria. The sample was

briefly

centrifuged,and the supernatantwasused in the PCR. PCRs were performed in 50 mM KCl-1.5 mM MgCl2-10 mM

Tris-HCl (pH 8.3)-0.01% (wt/vol) gelatin-100

puM

(each) deoxynucleoside triphosphate-50 pmol of each appropriate primer-1 ,ug ofT4 gene 32protein (Boehringer, Mannheim, Germany)-1.0 U of Taq polymerase (Cetus, Emeryville,

Ca-lif.). The sequences of the primers for toxin B were 5'-TAATAGAAAACAGTTAGAAA-3' (nucleotides410to429)

and 5'-TCCAATCCAAACAAAATGTA-3' (complementary tonucleotides 691to710),onthe basis of the sequence of the toxin Bgene (2),andresulted inaDNAfragmentof 301 bp. Samplesweresubjectedtoeither 35 or40cyclesof

amplifica-tion. Amplificationwas carried outin aDNA thermal cycler (Perkin-Elmer, Norwalk,Conn.),witheachcycle consistingof 1 minat 94°C, 1 min at 50°C, and 1 min at 72°C. Amplified

DNAwasdetectedby agarose gel electrophoresis (20) in the presenceof ethidium bromide. Confirmation of the

amplifica-tion products was obtained by using a digoxigenin-labeled (Boehringer) probeasdescribedbyWidjojoatmodjoetal.(27).

The probewassynthesized by PCR, with DNAofC.

difficile

ATTC 9689 used as atemplateand digoxigenin-dUTP added as alabel. Theprimersusedwere5'-GTCAGAGAATACTGT

AGTCG-3' (nucleotides508to527)and5'-TCCAATCCAAA CAAAATGTA-3'(complementarytonucleotides 691to710),

resulting ina digoxigenin-labeled probe with a length of 203

bp.

Detection of toxigenic C.

difficile

in spiked samples. Serial

10-fold dilutionsfrom overnightcultures of C.

difficile

ATCC

9689 were mixed with a negative stool sample, which was characterizedbya negative cytotoxicityassayresultandfrom whichno C.

difficile

wascultured. Thesespiked sampleswere processed bythe MIPAasdescribed above.Adirect PCRon these sampleswasalsoperformed.

RESULTS

Generation of MAbs.Oneagglutinating MAb (MAb 95.25; IgG3) that reacted with all tested C. difficile strains was obtained. These strains consisted ofboth toxigenic and non-toxigenicreferencestrains(ATCC 9689,ATCC43593, ATCC

43594,and ATCC 43596toATCC43603)andclinicalisolates

(Table 1). Furthermore,thereactivityof MAb 95.25wastested onothermicroorganisms whichareeithernormallypresentin the gut or pathogenic and which are the possible cause of

diarrhea (Table 1). Apart from agglutinationwith C.

difficile,

agglutination

wasalso found withC. sordelliiandClostridium

bifermentans.

TheMAb was not reactivein theimmunoblot.

Detection of toxigenic C.

dificile

in spiked samples. To establish thedetectionlimit of theMIPA,wecomparedMIPA with PCRperformed directlyon fecalsamples spiked with C.

difficile (Fig. 1).

Both

amplification

stepsconsisted of35

cycles.

Using

MIPA,wecould detect 3 x 10

toxigenic

C.

difficile

per

goffeces, whiledirectPCRwasnegativewith upto 1 x 10 C.

difficile

per g of feces. This means that at least a 104-fold increase in the detection level of toxigenic C.

difficile

was obtained.

Detection of toxigenic C.

dijicile

in clinical samples. We

processed 46 fecal samples. Of these 46 samples, 23 were positive in the cytotoxicity assay and

yielded

toxigenic

C.

difficile

inculture, 8

yielded toxigenic

C.

difficile

inculture but were negative in the cytotoxicity assay, 2 were

positive

for culture ofnontoxigenicC.

difficile

and

negative

inthe

cytotox-icityassay,and 13werenegativeinboth assays. Thefirstresults obtained with the 35-cycle MIPA without Chelex 100 were

disappointing

(see

the low sensitivitieslistedinTable

2).

Four

samples tested by MIPA were false

negative

in

comparison

with the results of the

cytotoxicity

assay, while nine

samples

were false negative in comparison with the results of culture fortoxigenicC.

difficile.

Toimprove the

sensitivity

and

specificity

of the

MIPA,

we increased the number ofcyclesfrom 35to40

(data

not

shown).

Although this improved the outcome of the assay, several amplification productswerevaguelyvisible in the agarose

gel,

which made the result difficult to interpret. After

adding

Chelex 100 and

using

the

40-cycle

MIPA,weobtained

bright,

clear-cutbands in the agarose gel

(Fig. 2).

The resultswere confirmedby Southern blot

hybridization.

The correlation of MIPAplusChelex 100 with culture for

toxigenic

C.

difficile

was excellent. Seven

samples

were

positive by

using

MIPA

plus

Chelex 100,while

they

were

negative by

the

cytotoxicity

assay.

Sampleswith

noncytotoxic

C.

difficile

remained

negative

in all MIPAs.

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TABLE 1. Agglutinationpatternsof severalClostridium strains with MAb95.25(mouseanti-C. difficile IgG3)a

Species No.of isolates Typeb Toxin

C.difficile 1 A +

C. difficile 1 B

C. difficile 1 A +

C. difficile 1 C +

C.difficile 1 D

C.

dificile

1 F +

C.

difficile

1 G +

C.

difficile

1 H +

C. difficile 1 I

-C. difficile 1 K

C. difficile 1 X

C. difficile 24 +

C.

difficile

24

C.difficile 8 NDC

C. bifermentans 3

C. sordellii 2

All strains described in the table were positive for agglutination. The

followingstrains werenegative for agglutination: Clostridium perfringens (n =

11), Clostridium butyricum (n = 1),Clostridium cadaveris (n = 1), Clostridium histolyticum(n=1),Clostridium innocuum(n =1), Clostridiumparaputnfucium (n = 1), Clostridium putnrucium (n = 1), Clostridium septicum (n = 1), Clostridium sporogenes (n = 1), Clostridiumsphenoides (n = 1),Bacteroides fragilis(n=5), Bacteroides vulgatus (n= 10), Bacteroidesthetaiotaomicron(n=

2), Bacteroides levii (n=5),Bacteroidescapillosus (n=3), Bacteroidesovatus(n =2), Bacteroides loescheii(n=2), Bacteroidescaccae(n = 1), Bacteroides bivius (n=1), Bacteroides merdae (n = 1), Shigellaflexneri (n = 3), Shigellasonnei(n

=2), Shigella boydii (n =3), Shigella dysenteriae (n = 3), Salmonellaserotype Durazzo(n= 1),Salmonella paratyphi (n=1), Salmonella typhimurium(n = 1), Salmonella serotype Heidelberg (n = 1), Salmonella abortusequi (n = 1), Salmonella serotype Derby (n = 1), Salmonella serotype Reading (n = 1), SalmonellaserotypeSchwarzengrund(n= 1), Salmonellaabortusbovis (n = 1), Salmonella serotype Nigeria (n = 1),Salmonella serotype Virchow(n = 1), SalmonellaserotypeAmersfoort(n=1),Salmonella serotypeGlostrup (n=1), SalmonellaserotypeTakoradi(n = 1), Salmonella serotypeNewport(n = 1), SalmonellaserotypeVirginia(n=1),Salmonella serotypeAmherstiana(n=1), Salmonella serotype Kentucky (n = 1), Salmonellaserotype Emek (n = 1), SalmonellaserotypeEimsbuettel(n= 1), Salmonella serotype Dublin(n = 1), Salmonella serotype Panama (n = 1), Salmonella typhi (n = 2), Salmonella enteritidis(n=1), Salmonella meleagridis (n = 1), Salmonellaanatum(n = 1), Salmonella Cerro(n=1), Escherichia coli serotypes 078 (n=1), 0127(n=1), 0166K?(n=1),0111K-(n=1),06K?(n =1), 021K2 (n=1),033K-(n=

1),023K18(n=1), 025 (n=1), 0128K- (n=1),0111K-(n=1),and

048K-(n=1), Campylobacterjejuni (n= 10), Yersinia enterolitica (n=3),Enterococcus

faecalis(n=4), andVibriocholerae(n= 1).

b Allstrains with knownserotype are ATCC strains.

cND,notdone.

-a

-b

c

-d

e--9

1

2

3

4

5

6

7

8M

B

-a

_

v=

~~~~~b

-c

-d

-e

-f

_g~~~~~~-12

3

4

5

6

7 8

M

FIG. 1. Detection limit ofMIPA (35 cycles) (A) compared with that of PCR performed directly on spiked fecal samples (B). (A) (MIPA).Lane1, positive control;lane2, negativecontrol;lanes 3to8, 10-fold dilutions ofspikedfecalsamples startingwith3 x 107inlane

3through3 x 102 in lane 8.(B)Samesamplesasdescribed forpanel A.Markers(lanesM)were asfollows:a,11,497 bp; b, 2,838 bp;c,1,700 bp; d, 1,159and1,093 bp;e,805bp;f,514bp;g,339bp.

also cross-reacts with C. sordellii and C. bifernentans (5). In 1981,Poxton andByrne (24)described theantigenic relation-ship of EDTA-extractedantigensfrom thesemicroorganisms; cross-reactionswerefound withantiseratovarious Clostridium

spp. However, antibodies reacting with C. difficile, C. irregu-laris,C. sordellii, andC. bifermentansdid not reactwith other Clostridium spp. Therefore, this group of Clostridium spp.

DISCUSSION

Direct PCR of feces is known tobeless sensitive than PCR ofpurified DNA because of the presence of inhibitory

com-poundsinfeceslikebilirubin, urobilinogens,andbile salts(28). To avoid these inhibitory factors, several investigators (4, 15, 18) perform PCR with stoolsamplesafter DNA extraction,a

method that is ratherlaborious. This canbe circumventedby magnetic immunoseparationof the microorganismsfrom the stool sample. To achieve magnetic immuno separation, we

madean agglutinatingMAbreactive with all known C.difficile serotypes(7, 8) andvarious clinical isolatescollectedfrom all

overThe Netherlands, andvarious strains obtainedfrom one

hospitalhavebeen shown to bedifferentbyrestriction restric-tion fragment length polymorphism analysis (30). Our MAb (MAb 92.25),however,wasnotspecificfor C.difficile alone;it alsoagglutinated C.sordelliiandC.bifermentans.For thepast several years, a commerciallyavailable agglutinationtest, the MicroScreen C. difficile latex slideagglutination test (Mercia Diagnostics, Guildford,UnitedKingdom),for the

demonstra-tion ofC.difficilehasbeenmarketed. LikeourMAb,thisassay

TABLE 2. Comparisonoftissueculturecytotoxicityassayand cultureofcytotoxicC. difficilestrains with MIPA

Percent

Assay Positive Negative

Sensitivity Specificity predictive predictive value value

TCCAV versus MIPA 82.6 91.3 90.5 84.0

for 35 cycles

TCCA versus MIPA 95.7 69.6 75.9 94.1

with Chelex 100 for 40cycles

Culture oftoxigenic 70.0 100 100 64.0

C. difficileversus MIPAfor 35cycles

Culture oftoxigenic 96.7 100 100 94.1

C. difficileversus MIPAwith Chelex 100 for40cycles

aTCCA,tissue culturecytotoxicityassay.

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A

b-

9-1 2 3 4 5 6 7 8 9 10 11 12 13 14

B

a

C

-1 2 3 4 5 6 7 8 9 10 11 12 13 14

FIG. 2. MIPA (40 cycles) plus Chelex 100 performed onclinical

fecalsamples.(A)Lane 1,positivecontrol;lane2, markers;lanes 3to

14, positive fecal samples. (B) Lane 1, negative control; lane 2,

markers;lanes 3to 14,negativefecal samples.Markers:a, 11,497bp;

b, 2,838bp;c, 1,700bp; d, 1,159and1,093bp, e,805bp, f,514bp;g, 339bp.

probablysharescommonantigens(24).Eventhoughthe MAb described here does not discriminate between toxigenic and

nontoxigenic isolates,itscross-reactivitydoesnotinfluence the

outcome of MIPA because theshortcomings of the MAb are

compensated forbythe selective amplification of the toxin B genespecific fortoxigenic C difficile.

The sensitivity, specificity, positive predictive value, and

negative predictive value of MIPA compared with the tissue culture cytotoxicity assay and culture of cytotoxic C. difficile strainsaregivenin Table2.Fromthe data in Table2,itcanbe concluded that the40-cycleMIPAwith Chelex 100is themost

sensitive assay for the detection ofcytotoxicC. difficile.These

findings agree withprevious results (28) that showed that the addition of Chelex 100contributestothesensitivityof PCRon fecalsamples.Theaddition of Chelex 100probablyleadstothe moreefficientlysisof C. difficile,theprotectionofDNAathigh

temperatures, and the inactivation of polymerase inhibitors

(6).

Sevensampleswerepositive byMIPAbutwerenegativefor

cytotoxininfeces,resultinginaspecificityof69.6% forMIPA

versusthe tissue culture cytotoxicityassay(Table 2).This may be explained bythe fact that, insome instances, nofree toxin

is present in feces, even though toxigenic C. difficle are

present. Recently, Gerding and Brazier (12) stated that

anti-biotic-associated diarrhea caused by C difficile can be

diag-nosedinapatientwith diarrhea after antibiotictreatmentonly

when toxins ortoxigenicC. difficile strains in stoolsamples,or

both, are detected. This supports the use of MIPAfor

diag-nosingantibiotic-associated diarrhea andquestions the useof the tissue culture cytotoxicityassayas thegold standard.

In an earlier study (29), we described the detection of

toxigenicC. difficile directlyafter cultureontheagarplateasa

sensitive and simple method.Eventhough colony blot hybrid-ization can be donereliably in anylaboratorybecause it does not need expensive equipment like a thermocycler and en-zymes, wethink that PCRwill become a generally accepted technique in the very near future. Kato et al. (18) detected toxigenic C.

difficile

in fecal samples by PCR by using an elaborate DNA purification method. However, the sensitivity of theassay described by that group was equal to that of the cytotoxicity assay (18). Gumerlock et al. (15) detected 103

toxigenic C. difficile infecal samples using an elaborate DNA extraction method and PCR. Our method displayed a lower detection limit than that described by Kato et al. (18). It was also assensitive as the method described by Gumerlock et al. (15) butwas much easier to perform and could be performed more rapidly. The time needed to perform the MIPA as described above, including sample preparation andanalyses, is less than7 h, which canbe shortenedconsiderably by using an improved system for detecting the amplicons. Although the presentstudy showed promising results, more elaborate clini-calstudies need to be performed toestablish the roleof MIPA amongroutine diagnostic methods.

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