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Serodiagnosis by passive hemagglutination test and verotoxin enzyme linked immunosorbent assay of toxin producing Escherichia coli infections in patients with hemolytic uremic syndrome

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Copyright (C 1994,American Societyfor Microbiology

Serodiagnosis

by Passive

Hemagglutination

Test and Verotoxin

Enzyme-Linked

Immunosorbent

Assay

of

Toxin-Producing

Escherichia

coli

Infections in Patients

with

Hemolytic-Uremic

Syndrome

SUMIO YAMADA,*AKEMI KAI,AND YASUO KUDOH

Departmenit of Microbiology, Tokyo Metropolitan Research Laboratory ofPuiblicHealthl, 3-24-1 Hyakunin-cho, Shinjuku-ku, Tokyo 169, Japani

Received 13 September 1993/Returned for modification 2 November 1993/Accepted 13January 1994

Eightcasesofhemolytic-uremic syndromeinwhichnopathogenswereisolatedwerediagnosedserologically by a passive hemagglutination assay and a verotoxin (VT; Shiga-like toxin) enzyme-linkedimmunosorbent assay(ELISA).Thepassive hemagglutination assayemployedformalinized sheep erythrocytessensitized with soluble native antigen or heat-treated antigen (lipopolysaccharide [LPSI) from Escherichia coli 026, 0111, 0128,and0157orflagellar antigenof nine different H serogroups of E. coli:H2, H7, H8, H10,Hl ,H12, H18, H19, and H25. All patients had antibodies against the native antigen and/or the LPS of E. coli 0157, but positive agglutination with H7 was observed only in one patient. In theVT-ELISA with plates coated with purified VT 1 or VT2, antibody againstVT 2wasobserved in theseraof five of sixpatients examined,but none of the patients possessed VT 1 antibody. These results indicate that the causative pathogen in these eight hemolytic-uremic syndrome cases is likely to be VT-producing E. coli 0157. The passive hemagglutination assaydescribed here is a verysensitive, simple,and rapidmethod. This assay ishighlyrecommended for the serological diagnosis ofVT-producingE.coli infections, particularly in patients infected byserogroup 0157 strains. Furthermore, theVT-ELISA is useful in studyingthe roleof VT in hemolytic-uremic syndrome.

Hemolytic-uremic syndrome (HUS) is manifested by the sudden onset of hemolytic anemia, thrombocytopenia, and acute renal failure about 10 days after the appearance of diarrhea and abdominal cramps. Although various bacterial and viralagentshad been reportedasthecausativepathogens (15), Karmali et al. (9-11) reported that a verotoxin (VT;

Shiga-like toxin)-producing E. coli (VTEC) strain, 0157:H7,

was detected frequently in the stools of patients with HUS. Sincethen, the correlation between HUS andVTEChasbeen wellstudied in America, Canada, and Europe (11, 12, 16, 17). InJapan,theincidence of VTECinfection has beenincreasing sinceanoutbreak of VTEC 0157:H7 infection at a kindergar-tenin 1990, but thecorrelation between HUS andVTEC has notbeenclearly demonstrated. The detection of VTECinthe stools of patients with HUS is the most definitive diagnostic criterion. Itcansometimes beverydifficulttodetectVTEC, as theorganismcanbe recovered from stoolsonly forashorttime after diarrhea. HUS appears about 1 week after the onsetof diarrhea, atwhich time the pathogen usuallyhas disappeared from the stool. Also, treatment with antibiotics has been startedbefore HUSappears.On the otherhand,HUSpatients produceahigh level of antibodyresponse tothepathogen(13), and two studies (1, 3) have exploited this observation in the serodiagnosis of HUS.Wehave also reported the development of bothapassive hemagglutination (PHA)assayusing forma-linized sheep erythrocytes (FSRBC) sensitized with soluble native antigen or heat-treated antigen (lipopolysaccharide [LPS]) of VTEC 0157 and a VTenzyme-linked immunosor-bent assay(ELISA) (21). In ordertostudy the utilityofthese

*Correspondingauthor. Mailing address: Department of Microbi-ology, Tokyo Metropolitan Research Laboratory of Public Health, 3-24-1, Hyakunin-cho, Shinjuku-ku, Tokyo 169, Japan. (Phone): 03-3363-3231. (Fax): 03-3368-4060.

assays for the diagnosis of VTEC infection, we examined diarrhealpatients in the outbreak whichoccurredat a kinder-garten in 1990 and in three sporadic cases which occurred in 1991 and 1992. ThePHA assay showed that49(74.2%) of66 patients of the kindergarten outbreak and all of the sporadic case patients had antibodies to both or either the native antigenandthe LPS of 0157. In addition,anti-VT 2antibody detected by the VT-ELISA was observed in the sera of 17 patientsfrom the outbreak. Inthis study,wefurther evaluated the utility of these assays foreight HUS patients in whom no pathogenswere detected.

MATERIALS ANDMETHODS

HUS patients. The eight HUS patients in this study were identifiedatsixdifferenthospitals from May 1990toMay1993. All patients (A through H) are children 2 to 7 years old, as shown inTable 1. Allpatients presented tomedical clinicsat the onsetofgastrointestinalsymptoms and were subsequently hospitalized about 1 week after the onset. The most common symptoms included abdominalcramps, bloodyorwatery diar-rhea, and fever. Vomitingwasobserved in threepatients (A,B, andE). Bacterial examinations of fecal specimens from these patientswerecarriedout atthe hospitals with sorbitol IPA-bile salt agar(Kyokutou Seiyaku, Tokyo, Japan) and other media. VTEC or otherpathogenswere notisolated from these stools. Onepatient (D)wasculturepositivefor E. coli0128,but the isolate didnotproduceVT.Hematological investigationswere alsocarriedout atthe hospitals. Mostofthe patients suffered from hemolytic anemia, thrombocytopenia, and acute renal failure. Theywere diagnosed as having classical HUS. Their hemoglobinlevels were 5.8 to 8.4g/dl; haptroglobinlevels,6.8 to 21.8mg/ml; plateletcounts, 0.9 x 10to4.8 x 10 per mm; blood urea nitrogen levels, 39 to 132 mg/dl; and creatinine levels, 1.2 to 4.5 mg/dl.

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TABLE 1. Antibody responses to antigens and VTs of E. coli in HUS patients Antibodyresponsea

Patient

Wk after PHAtiter with FSRBC sensitized with:

A405'

in ELISA coated with:

onsetof Flagellar

Age diarrhea Nativeantigen LPSantigen agen

Designation (yr) Sex Va 1 VT2

026 0111 0128 0157 026 0111 0128 0157 H7 Othersc

A 2 F 1 -d 300e - 300 - 0.078 0.212

6 900 - 900 - 0.088 0.246

B 4 F 1 8,100 - 8,100 900 0.102 0.207

3 2,700 - 2,700 300 0.095 0.215

4 300 - 300 100 0.075 0.100

10 - - 300 - 100 - 0.103 0.113

C 7 F 1 900 - - 2,700 - 0.094 0.186

2 2,700 - - - 2,700 - 0.100 0.210

D 2 M 13 - 300 - 300 - 0.088 0.094

E 7 M 2 2,700 - - - 300 - 0.141 0.283

6 900 - 100 - 0.085 0.156

F 4 M 1 - 900 - 100 - Notdone Notdone

6 - 300 - - Notdone Not done

G 3 M 4 100 - - - Not done Not done

6 100 - - - Not done Not done

H 7 M 1 900 - 300 - 0.186 0.225

2 900 - - 300 - 0.163 0.205

Control

Patient -f 2,700 - 2,700 - 0.094 0.241

Patient2f 2,700 - 900 - 0.077 0.218

20healthy adults - - - 0.040-0.023 0.038-0.010

10healthychildren - 0.038-0.035 0.037-0.017

Serumsampleswerediluted more than100-foldfor the PHA test and1,000-foldfor the ELISA. Avalue of 0.2 wasdeterminedtobethe minimumindicationof thepresenceofantibody.

cH2, H8, H10,Hit,H12, H18, H19,and H25.

d-,Nonagglutination in 100-fold dilutionof serumsample.

e Data are thereciprocalsof thehighestdilution which causedagglutination.

fVTEC0157:H7 wasisolated, and thepatientdevelopedHUS(21).Serum was taken 5days after theonsetofdiarrhea.

Exceptforonepatient (B), allpatients recoveredafter2 or 3 months oftreatmentwith aspirin, dipyridamole, heparin, or

dialysis. In patient B, necrosis occurred in the colon

region,

and the colon wasremoved.

Serum samples. Serum

samples

were collected from the

eightpatients atthe acuteorconvalescent

phase.

After exam-ination ofhematological parameters at the

hospital

laborato-ries, these serum sampleswere transferred to our

laboratory

andstored at -20°Cuntil use.

Sera from 20 healthy adults and 10

healthy

children were usedas negative controls, and two of theHUS

patients

from the kindergarten outbreak

(21)

from whom fecal VTEC 0157:H7wasisolatedwere used as

positive

controls.

Bacterial strains. For

preparation

of native

antigen

and LPS, four common VTEC 0 serogroups, strains TEC100

(VTEC 0157:H7), TEC182

(VTEC

026:H11),

TEC205 (VTEC 0111:NM), and TEC347

(VTEC

0128:H2),

were used.

Forpreparationofflagellarantigen,strains TEC347

(0128:

H2), TEC100 (0157:H7), TECO03

(02:H8),

TEC112

(0148:

H10),TEC182

(026:H11),

TECO43

(09:H12),

TEC135

(017:

H18), TEC224

(OUT:H19),

and

TECO45

(015:H25)

were used.

Purified VT 1 and VT 2. VT 1 and VT 2were

purified

from cultures of

Shigella

dysenteriae type 1 and VTEC

0145:NM,

respectively, by the following procedures. Each strain was inoculated intoTrypticasesoybroth

(BBL)

andincubated for 18hwithconstantshaking.Sterile filtratewasmixed withsoiid

ammonium sulfate upto a 70% saturation level. The

precipi-tate was

suspended

in 0.05 M

phosphate

buffer

(pH

6.0)

and

dialyzed against

the same buffer. The

dialyzed

crude

sample

wasloadedonto a

DEAE-Sephacel

column

(Pharmacia)

equil-ibrated with 0.05M

phosphate

buffer

(pH

6.0)

andeluted with alinear

gradient

of 0to0.5 M NaCl in 0.05

phosphate

buffer,

pH

6.0.Fractions

showing toxicity

inthe Vero cell assaywere

pooled

and

dialyzed

against

0.005 M

phosphate buffer,

pH

6.0. The

dialyzed

sample

was

applied

onto a column of PBE

(Pharmacia)

equilibrated

with 0.025 M

imidazole-HCl,

pH

7.4.

Polybuffer

74

(Pharmacia)-HCl

(pH

6.0)

wasusedtoelute VT 1from the PBE

column,

and

polybuffer-HCl

(pH

3.6)

wasused toeluteVT2. VT1waseluted ina

peak

at

pH

6.0,

whereasVT 2waseluted in a

peak

at

pH

4.0.Toxicfractionswere

pooled,

andsolid ammonium sulfatewasadded tothe

pooled

sample

to 1.7 M. The mixture was

applied

to a fast

protein liquid

chromatography

system

(FPLC;

Pharmacia) equipped

with a

phenyl-Sepharose

CL 4B

(Pharmacia)

columnwhichwas

equil-ibrated with 1.7 M

(NH4)2SO4-0.05

M

phosphate

buffer

(pH

6.0),

and the toxin was eluted with a linear

decreasing

salt

gradient

(1.7

to 0

M)

in 0.05 M

phosphate

buffer, pH

6.0.

Finally,

thefractions from

hydrophobic

chromatography

show-ing

VT

activity

were

directly

applied

toacolumnof TSK

gel

G 3000SW

(Toso Co.,

Tokyo,

Japan) equilibrated

with 0.05 M

phosphate

buffer

(pH

6.0)

andfractionated inanFPLCsystem.

The

purified

toxin from cultures of each strain gavea

single

band onconventional

polyacrylamide

gel

electrophoresis

(5).

In the

Ouchterlony

gel

double-diffusion test between the

purified

toxin(s)

and

anti-Shiga

toxin

(22)

or anti-VT 2

(23)

serum

(gifts

from Y.

Takeda,

School of

Medicine, Kyoto

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University),

thetoxin from S.

dysenteriae

1formeda

precipitin

line withthe

anti-Shiga

toxinserumbutnotwith the anti-VT 2

serum. Jo contrast,the toxin from VTEC 0145:NM formed a

precipitn.-

line with anti-VT2serumbutnotwith the

anti-Shiga

toxin serum.

Preparation

of native

antigen

and LPS. Strains grown in

Trypticase

soy broth

(BBL)

were cultured on nutrient agar

plates

at

37°C

and incubated

overnight.

The

organisms

were harvested in saline and

disrupted

withaFrench press

(Ohtake

Co.,

Ltd., Tokyo,

Japan)

at apressure of

1,000

kg/cm2.

After

centrifugation

at

23,000

xg for 20 min, the supernatant fluid

wasfiltered

through

a membrane

(pore size,

0.45 ,um; Milli-pore

Corp., Bedford, Mass.).

One filtratewasused for native

antigen,

andanother filtrate heated at

121°C

for 30 min inan autoclave and

centrifuged

at

1,500

xgfor 20 minwasused for heat-treated

antigen (LPS).

Flagellar antigens.

Nine kinds of

flagellar

antigens

were

partially

purified

from the culture grown in

Trypticase

soy broth

by

the method of Ibrahim etal.

(8).

FSRBC sensitized with

antigen(s).

FSRBC

(Japan

Biotest

Lab, Tokyo, Japan)

werewashed four times and

resuspended

ataconcentration of10% insaline.An

equal

volumeof tannic acid

(0.1

,ug/ml; Japan

Biotest

Lab)

was

added,

and the mixture

was incubated in a

37°C

waterbath. After 30min, cellswere washed three times with cold saline and

resuspended

at a concentration of10%.

Antigens

tobe testedwereaddedtothe FSRBC at a concentration of 100

,ug/ml.

The mixture was incubatedat

37°C

for 30 min and lefttostandat

4°C overnight;

then the FSRBC were washed three times with saline to

remove unbound

antigen.

The sensitized FSRBC were

sus-pended

at a concentration of 10% in saline

containing

1%

sodium azide and storedat

4°C

untiluse.Control FSRBCwere

prepared

in thesameway except that the addition of

antigens

wasomitted. Priortouse, the FSRBCstockswere dilutedto a concentration of 1% in saline

containing

0.1% bovine serum

albumin

(Sigma)

and0.01% Tween 80

(Sigma).

PHA. The PHA test was done in a microwell

plate

with

V-shaped

wells

(Greiner),

as described

previously

(21).

The

PHAtiterofserum

against

each

antigen

was

expressed

asthe maximum dilution that showed

agglutination.

Treatment of

patient

serum with 2ME. To

investigate

the

immunoglobulin

class of the antibodies revealed

by

reactions in the PHA assay and

VT-ELISA, positive

sera of

patients

were treatedwith

2-mercaptoethanol (2ME) (6).

Serum

sam-ples

(0.1 ml)

from

patients

werediluted 10-fold in

saline,

and

an

equal

volume of0.2 M 2ME

(Wako

PureChemical

Indus-tries,

Tokyo, Japan)

wasadded. Afterincubationat

37°C

for 1

h,

2 ml of cold acetone was added to remove the 2ME and

precipitate

the serum

protein.

The mixture was

centrifuged,

and the acetone was removed with an

aspirator.

After the

acetone treatmentwas

repeated

once, the

pellet

wasresolved

in 0.1 ml of saline and

subjected

to the PHA assay and VT-ELISA.

VT-ELISA. Asdescribed

previously (21),

the VT-ELISAwas carried out

according

to the recommended procedure for ELISAmate

(Kirkegaard

&Perry

Laboratories),

using purified

VT 1 andVT 2.

RESULTS

Specificity

of FSRBCsensitizedwithantigen(s).The

speci-ficity

oftheFSRBC sensitized with

antigen(s)

wastestedbythe PHA assay,

using specific

antisera to the respective 0 or H

antigens

of E. coli

(Denka Seiken,

Tokyo, Japan). FSRBC sensitized with each of the native

antigens

or LPS of four

predominant

VTEC 0 serogroups

(026,

0111,

0128, and

0157) or the flagellarantigen of nine common H serogroups (H2, H7, H8, H10, Hll, H12, H18, H19, and H25) aggluti-nated only with the homologous antiserum. To assess the utility of the PHA assay, we measured the reactivity of 30

negativecontrolserumsamplestothe variousantigens. When twofold dilutions of the negative controlsera in the range of 1:10to1:640weresubjected to the PHA assay, low PHA titers (1:10 to 1:80) against antigens 026, 0157, H2, Hll, and H25 were observed insome serum samples. As dilutions of 1:100 and greater didnotresult inpositive reactions with any of the antigens tested, this dilution was used as the initial dilution of serum.

Reaction of sera from HUS patients with native or LPS

antigen and flagellins from E. coli. The results of the PHA assay are summarized in Table 1. All serum samples from patients takenattheacuteorconvalescentphase reacted with the 0157 native and LPSantigens, except sera from patients F and G taken 4 or 6 weeks after the onset ofdiarrhea; these reacted only with 0157 native antigen. None of the sera reacted withantigensof the otherVTEC serogroups, e.g., 026,

0111, and 0128. The PHA titersagainst 0157 native antigen and LPS variedwidely in individual patients. However,mostof thepatients had acquired significantly higher levels of antibod-ies against native or LPS antigen in the early stage of the infection. The PHAtiters tendedtofallrapidly after the acute

phase of thedisease, buttheywere sometimes detectable 10 weeks after theonset ofthe disease, as in patients B and D.

Furthermore, these antibodieswere inactivatedby treatment with2ME.

PHAtestingfor antibodiesagainst nine commonVTEC H

antigensshowed thatonlyonepatient,caseB, had antibodies

againstH7attheacutephase.The PHA titer ofserumsamiples taken at 1 and 2 weeks after the onset of illness was 1:900. After 4weeks,the titerwasbelow 1:100. These antibodies were also inactivatedbytreatmentwith2ME.

VT antibodies detected in patients' sera by VT-ELISA. In

preliminary experiments, negativecontrolseradiluted 100-fold gavean

A405

of 0.62to3.11inwells coated withVT 1 or VT2 andanA405of 0.24to0.35 inwells withoutantigens.Since the absorbance in bothantigen-coatedanduncoated wells with the

1,000-fold dilution of these serawas in the range of 0.01 to

0.04, thisdilution wasused inVT-ELISA.

On the basis of results of the VT-ELISA testofserafrom

patients in the outbreak causedby VTEC 0157:H7 (21), an absorbance of 0.2wasestablishedasthe cutoff valuetoindicate the presence of anti-VTantibody. An absorbance of 0.11 to 0.19 was considered a suspicious positive reaction, and that below0.1 wasnegative.

Six serum samples of from the eight HUS patients were tested for anti-VT antibody. As shown in Table 1, ELISA

readings for VT 2 showed that the absorbance of serum

samples from patients ranged from 0.094 to 0.283. The sera from fivepatientsin whomanabsorbance of above 0.2 (cutoff

value) was observed indicated the presence of anti-VT 2

antibody.

Antibody against VT 2 was foundmainly in serum

samples

taken within 3 weeks of the onset of illness. VT 2

antibodyalsotendedtofallrapidlyaftertheacutephase of the

illness, although the serum sample from patient A taken 6 weeks after the onset of diarrhea still was positive for VT 2

antibody.Thesepositivesera werealso treatedwith 2ME, and

nonspecific reactions showing an absorbance of >2.0 were observed in all samples tested. On the other hand, ELISA results forVT 1 showedabsorbance ofserumsamples ranging from 0.075 to 0.186. None of the sera tested showed an absorbancehigherthan the cutoff value.However, a suspicious

positive

reactionwasobserved intwopatients'sera(patientsE

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and H) within 2 weeks after the onset of the illness. An absorbance of 0.141 for patient E and one of 0.186 or 0.163 for patient H were noted.

DISCUSSION

Investigations (19) of HUS in Japan carried out in 1991 showed that the incidence of HUS has been increasing since theoccurrenceofanoutbreak of VTEC 0157:H7 infection at akindergarten in 1990. Approximately 100 HUS cases per year have been confirmed, and about 76% of these presented in

typicalfashion. Amongthese cases, gastrointestinal symptoms werethe first signs of the disorder. In 43% of those cases, the causative pathogen was identified. E. coli was isolated from 76% of those stool samples in which any pathogen was identified. However, only14%of these pathogens were VTEC. Itis difficult to detect VTEC or any other pathogens in many cases of HUS. In the eight HUS cases in this study, no enteropathogensweredetected insevenpatients. Although E. coli 0128 was isolated from one patient, this strain did not produceVT.Therefore, the development of other methods for thediagnosis of the causative pathogen in HUS is desirable.

In order to diagnose VTEC infections serologically, we previously developed a PHA assay for the detection of anti-bodiesagainst native antigen, LPS, and the flagellar antigen of VTEC 0157:H7, and aVT-ELISAtodetect VT 1 and VT 2. Since theutilityof these assayswasstudied withserumsamples from diarrheal patients infected with VTEC 0157:H7 (21), we further assessed the usefulness of these assays by examining

eight HUS patients in whom the causative pathogen wasnot isolated. In addition, for the application of these assays to HUS, three predominant VTEC 0 serogroups (026, 0111, and0128)andeight commonHserogroupsofE.coli(H2, H8, H10, H11,H12, H18, H19, andH25)wereaddedtothestudy, becauseVTEC strainsbelongingtovarious 0orHserogroups apart from 0157:H7 have been isolated from patients with HUS (10, 17).

The results showed that allpatients had antibodies against the native antigen and/or the LPS of E. coli 0157 in serum

samplestakenattheacute orconvalescent stage of theillness,

but none of the sera reacted with the 0 antigens of other VTEC serogroups, suchas026, 0111,and 0128. InthePHA testfor the ninecommonHantigens, onlyonepatient(B)had antibodies against H7 in acute-phase serum samples. In the

VT-ELISA, antibodies against VT 2 were observed infive of sixpatients examined.

With regard to serodiagnosis of HUS, Chart et al. (3,

4)

developeda0157LPS-ELISA,andrecentlyBitzan and Karch

(1)reportedanindirecthemagglutinationassaythatuses0157 LPS-coated sheep erythrocytes. In both assays, antibody against 0157 LPSwasobserved in the seraof HUS

patients.

Those studies also recommended the detection of antibodies

againstnative antigen orLPS of E. colibymeans ofELISA,

indirecthemagglutination orPHAfor thediagnosisof VTEC infections when fecal VTECorVTisnotdetectable. We also tried to detect antibody against

flagellar antigens

of VTEC. However, the results of the PHA test for antibodies against

ninecommonVTECHantigensshowed thatonlyoneofeight

patients had antibodies against H7 in acute-phase serum samples. In the remaining seven cases, antibody against H7 antigen could not be detected, although most ofthe VTEC 0157 strains isolated frompatientswith

hemorrhagic

colitisor HUS are motile and possess the H7 antigen (12). In our previousworkregardingthe outbreak of VTEC 0157:H7(21), antibody againstflagella antigenofH7 was notdetected in16

hospitalized patients orin 50outpatients. Chartetal.

(3)

also

could not detectantibodies against H7 in serum samples of 13 HUSpatients in spite of their higher titers of antibody against 0157 LPS. It is not clearwhether the inability to detect the antibody against H7 is due to the methods used or to other factors. However, a possibility exists that some E. coli 0157 strains in human infection possess cryptic H7 or nonimmuno-genic H7. In either case, our PHA assay to detect antibody againstflagella antigen may not be suitable for the serological diagnosis of VTEC infection.

Intheserodiagnosis of VTEC infection with the PHA assay, there is a problem in that the LPS of E. coli 0157 shares commonantigenic epitopes with the LPS of certain serogroups ofBrucella abortus(18),Yersiniaenterocolitica (20), Salmonella spp. (20), and Vibrio cholerae non-O-1 strains (7). Antibodies directed against these organisms may give nonspecific reac-tions in the assay. Our data (unpublished data) showed high levels of cross-reaction between FSRBC sensitized with 0157 native antigen or LPS and antibodies of B. abortus and Salmonella serogroup 030, butnocross-reactionwasdetected with Y enterocolitica serotype 09 or V. cholerae non-0-1 serogroup Hakata. Since the symptoms ofB. abortus infection are quite distinct from those of HC or HUS, misdiagnosis resulting from the cross-reaction in the serum seems unlikely.

Similarly, Salmonella serogroup 030 is very rarely isolated from patients with diarrhea. On the other hand, nonspecific reactions with test serain thePHAassay couldbeeliminated by the dilution of sera; therefore, the 100-fold dilution was considered appropriate for the PHAassay.

ThePHAtitersagainst 0157 native and LPS antigens varied widely in individual patients. However, most of the patients

acquired significantly high levels of antibody tobothantigens in the early stages ofinfection. ThePHAtiters tended to fall rapidly after the acute phase of the disease, but they could sometimes be detected even 10weeks after the onset of the illness. Similar trends in the antibody level of patients with HUS wereobserved in other studies(1, 3). Bitzan and Karch (1) indicated that this rapid fall in antibody titertothe E.coli 0157 LPS may be duetothe short half-life ofimmunoglobulin

M (IgM). To study the immunoglobulin class related to

positive reactions in the PHA assay and VT-ELISA,positive

seraof HUS patientswere treated with 2MEbecauseIgM in serum is segregated from the 19S to the 6.5S subunit by

reductionthroughasulfhydratereagentsuchas2ME(6).IgM

antibody treated with 2ME becomes nonreactive. The treat-mentof the antibodies with 2ME in thisstudy suggestedthat the immunoglobulin class of the antibodies detected in the PHAassayand VT-ELISA is IgM.

ByusingtheVT-ELISA,anantibodyresponsetoVT 2 was observedin five of sixpatients' sera.This result indicates that VT-ELISA is useful as one method for the serodiagnosis of VTEC infection. Karmalietal.(11) reportedthat4of 15cases ofsporadicHUS and 2

patients

with

uncomplicated

diarrhea whoweresiblingsof HUSpatientshadneutralizingantibodies

against the VT by Vero cell assay. VT-ELISA is

highly

sensitive and more convenient than the Vero cell assay to detect neutralizing antibody, but the usefulness of the VT-ELISA as a diagnostic tool may be limited to the general

laboratory with access to pureVT. However, theVT-ELISA can be used to examine experimentalverotoxemiain

rabbits,

monkeys,orotheranimalsand toclarifytheserologicalfactors involved in HUS.

In the VT 1-ELISA, a suspicious positive reaction was observedintheserumsamplesfromtwopatients.

Although

we

usedanti-humantotalimmunoglobulinantiserumas a second-ary antibody, the use of fractionated immunoglobulin anti-serum may increasethe specificity of the VT-ELISA.

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In recentwork reportingon the antibody against VT (2), a non-specific neutralizing effect of normal human sera against VT 2 and the non-IgG fraction was noted, suggesting the prcsenre of a VT 2 inhibitor. In VT-ELISA, a nonspecific reaction was also observed in some serum samples from healthy individuals when the samples were diluted 100-fold. This reaction could be excluded by a higher dilution (1,000-fold) of serum. However, it is unclear whether these non-specific reactions are caused by some factor(s) in normal human sera. As onepossibility, globotriosyl ceramide, one of theplasma-bound glycolipids, may be involved in these reac-tions. It has been consistently extracted from normal human plasma and recognizedasa receptorofVT(s) (14).

ThePHA assay described here is simple to perform and was sensitive enough to detectantibodies against VTEC in patients with HUS. We highly recommend the PHA assay for the serological diagnosis of VTEC infections caused mainly by serogroup 0157. This assay is basically the same as that described by Bitzan and Karch (1), except for the use of FSRBC; however, the sensitized FSRBC are stable for 6 months at5°C, the PHA canbe performed immediatelyafter theserumsamplesareobtained,and the resultsareavailable in 2 to 3h.Furthermore, the VT-ELISA can be used to examine theVTin HUS and tostudy the role of VT in HUS.

ACKNOWLEDGMENTS

WethankMasakoKaminaga, Shouwa GeneralHospital; Hiroyuki Seki andShigeru Tomozawa,School ofMedicine, GunmaUniversity; Kyoko Hoshino, Tohou University Hospital; and Kikuo lidaka, KitazatoUniversityHospital,forprovidingclinical andhematological information and serumsamplesfrom HUSpatients.

REFERENCES

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5. Davis, B. J. 1964.Disc electrophoresis. II.Methods and applica-tion to humanproteins.Ann. N.Y.Acad. Sci. 121:404-427. 6. Deutsch, H. F., and J.I. Morton. 1957. Dissociation of human

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Medical Science Program on Cholera and Related Diarrheal Diseases.

8. Ibrahim, G. F., G. H.Fleet,M.J.Lyons, andR.A.Walker.1985. Methods for isolation ofhighlypurified Salmonella flagellins. J. Clin. Microbiol. 22:1040-1044.

9. Karmali, M. A. 1989.Infection byverocytotoxin-producing Esch-erichiacoli.Clin. Microbiol. Rev. 2:15-38.

10. Karmali, M. A., M.Petric, C. Lim, P. C.Fleming,G. C.Arbus, and H. Lior. 1985. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Esche-richiacoli.J.Infect.Dis. 151:775-782.

11. Karmali, M. A., B. T. Steel, M. Petric, and C. Lim. 1983. Sporadic casesofhemolyticureamic syndrome associated with fecal cyto-toxin andcytotoxin-producing Escherichia coli in stools. Lancet i:610-620.

12. Kleanthous, H., H. R. Smith, S. M. Scotland, R. J. Gross, B.Rowe, C. M. Taylor, and D. V. Milford. 1990. Heamolytic ureamic syndromesin the BritishIsles, 1985-8: associated with verocyto-toxinproducing Escherichiacoli.2.Microbiologicalaspects.Arch. Dis.Childhood 65:722-727.

13. Levin,M., M. D. S.Walters, and T. M. Barratt. 1989.Hemolytic uremic syndrome. Adv.Pediatr. Infect.Dis.4:51-81.

14. Lingwood, C. A., H.Law, S.Richardson, M. Petric, J. L. Brunton, S. D. Grandis, and M. Karmali. 1987. Glycolipid binding of purified andrecombinant Escherichiacoliproduced verotoxin in vitro.J.Biol.Chem. 262:8834-8839.

15. Milfold,D.V., C. M.Taylor, B.Cuttridge,S. M.Hall, B. Row, and H. Kleanthous. 1990. Haemolytic ureamic syndromes in British Isles1985-8:association withverocytotoxinproducing Escherichia coli.1.Clinical andepidemiologicalaspects.Arch.Dis.Childhood 65:716-721.

16. Riley, L. W. 1987.Theepidemiologic, clinical,andmicrobiologic featuresofhemorrhagic colitis.Annu. Rev.Microbiol. 41:383-407. 17. Scotland, S. M., B. Row, H. R. Smith, G. A.Wilshaw, and R. J. Gross.1988.Verotoxin-producingstrains ofEscherichiacolifrom children with HUS and theirdetectionby specific DNAprobes.J. Med. Microbiol. 25:237-243.

18. Stuart, F. A., and M. J. Gorbel. 1982.Identificationofaserological cross-reaction between Brucella abortusand Escherichiacoli.Vet. Rec. 110:202-203.

19. Takeda, T., Y. Oku, S. Yamasaki, and Y. Takeda. 1991. Examina-tion methods for the detection of verocytotoxin produced by hemorrhagicEscherichia coli and antibody in serum against its organism.Clin. Microbiol. 18:77-84. (In Japanese.)

20. Thomas, L. V., R.J. Gross,T.Cheasty,C. R.Shipp, and B. Rowe. 1983. Antigenic relationships among type strains ofYersinia en-terocolitica and those of Escherichia coli, Salmonella spp., and Shigellaspp. J. Clin. Microbiol. 17:109-111.

21. Yamada, S., S. Matsushita, A. Kai, M. Sasaki, A. Tsuji, T. Kanemitsu, N. Yamashita,E.Anzai, and Y. Kudoh. 1993. Detec-tion ofverocytotoxinfrom stool andserologicaltesting ofpatients with diarrhea caused by Escherichia coli 0157:H7. Microbiol. Immunol.37:111-118.

22. Yutusdo, T., T. Honda, T. Miwatani, and Y. Takeda. 1986. Characterization ofpurifiedShiga toxin fromShigelladysenteriae 1.Microbiol. Immunol. 30:1115-1127.

23. Yutsudo, T., N. Nakabayashi, T. Hirayama, and Y. Takeda. 1987. Purification andsomeproperties ofaverotoxinfromEscherichia coli 0157:H7 that is immunologically unrelated to Shiga toxin. Microb. Pathog.2:339-349.

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