Vol. 13, No. 1JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1981, p. 106-1140095-1137/81/010106-09$02.00/0

Improved Serodiagnosis of Salmonella Enteric Fevers by anEnzyme-Linked Immunosorbent Assay

WILLIAM J. BEASLEY,* SAM W. JOSEPH,t AND EMILIO WEISSNaval Medical Research Institute, Bethesda, Maryland 20014

The value of the enzyme-linked immunosorbent assay (ELISA) for the serolog-ical diagnosis of typhoid or paratyphoid fevers was tested with a collection of fivesets of sera from 23 individuals who had no history of Salmonella infection froma nonendemic area (set 1) and from 143 patients from a highly endemic area (sets2 to 5). The test was performed with a crude cell envelope fraction derived fromSalmonella typhi. On the basis of results with sera in set 1, titers >160 (inverseof serum dilution) for immunoglobulin M (IgM) or >500 for IgG were regarded assignifying a specific antibody response. These titers were occasionally exceeded insera in set 2, derived from patients with no clinical or laboratory evidence ofSalmonella infection. In presumptive cases of typhoid or paratyphoid fevers(culture positive and agglutination negative or vice versa; 13 patients, set 3, and26 patients, set 4, respectively), IgG antibodies were encountered more frequentlythan IgM, but some sera were negative in each group. In 81 confirmed cases(culture and agglutination positive, set 5) IgG antibodies were detected in at leastone of the paired sera from all patients, and the IgG titers were higher (median3,500) than those of the IgM antibodies (median 600). There was no evidence ofa significant difference in IgM or IgG titers between acute- and convalescent-phase sera. The titers of 10 paired sera from patients from whom S. enteritidis,bioserotype paratyphi A, was isolated were somewhat lower than those of 84patients with S. typhi isolation (serogroup D). There was some correlationbetween O antigen agglutination and IgM enzyme-linked immunosorbent assay,but otherwise the titers in the two serological tests varied independently. Of 11patients in set 3 (2 patients with serogroup B agglutinins omitted), 1 belonged toblood group O, in contrast to 54 group O among 107 agglutination-positivepatients, a distribution not necessarily reflected by the enzyme-linked immuno-sorbent assay. This study has provided good evidence that the enzyme-linkedimmunosorbent assay can serve as a valuable aid to the diagnosis of Salmonellainfection in an endemic area, as a substitute for the agglutination test.

The value of the Widal test (bacterial agglu-tination) for the diagnosis of febrile illnesses dueto Salmonella typhi and other Salmonella hasbeen analyzed by several investigators. In someendemic regions where laboratory facilities arequite limited, bacterial agglutination tests areamong the few available to clinicians who mustdifferentiate enteric infection from febrile ill-nesses due to other bacteria, viruses, or animalparasites (1, 14). However, it is recognized thatagglutination tests have serious shortcomings.Discrepancies in results obtained in differentlaboratories or in the same laboratory with prep-arations from different sources have been un-comfortably high (8, 9). In a significant numberof patients with otherwise well-documented ty-phoid fever, development of agglutinins has not

t Present address: Naval Medical Research and Develop-ment Command, Bethesda, MD 20014.

been demonstrated (14, 25, 28). On the otherhand, agglutinins against S. typhi are often ob-tained in response to nontyphoid Salmonellainfections (21, 24, 25) and sometimes in diseasesnot attributable to Salmonella (20). The mostimportant shortcoming, however, as the test isusually performed in the clinical laboratories, isthat it is primarily a test of immunoglobulin M(IgM) antibodies (5, 6, 13). Hemagglutinationtests, which offer several advantages over bac-terial agglutinations in terms of sensitivity andversatility (19), often depend on IgM antibodies(2, 31).Carlsson et al. (3, 4) and Svenungsson et al.

(29) applied the enzyme-linked immunosorbentassay (ELISA) to the titration of antibodies toSalmonella O antigens. These authors testedpurified lipopolysaccharide antigens derivedfrom several serogroups against specific rabbit

106

on January 3, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

SERODIAGNOSIS OF ENTERIC FEVERS BY ELISA 107

antisera and human sera with O agglutinationtiters >160 (inverse of serum dilution) for sero-groups D or B. Their results indicated that theELISA correlated well with the agglutinationreaction, was approximately 100-fold more sen-sitive, was highly reproducible, and displayedsome serogroup specificity. In a more limitedstudy of the ELISA with Salmonella antigens,Sippel et al. (26) demonstrated that a cell enve-lope fraction (CE) derived from S. typhi was asatisfactory antigen. This antigen (12) is rela-tively crude and contains lipopolysaccharide andprotein, but relatively low levels of flagellar com-ponents. Encouraging results were obtained withsera from proven typhoid and paratyphoid Acases that had reacted weakly in the agglutina-tion test.The availability of a serum collection from

patients from a highly endemic area provided uswith an opportunity to determine the value ofthe ELISA with a single crude antigen, preparedas described by Sippel et al. (26). The resultsclearly indicate that the ELISA eliminates mostof the shortcomings often encountered in theagglutination test.

MATERIALS AND METHODSOrganisms. The source and maintenance of S.

typhi strain T68, a human isolate, used in this studyhave been described elsewhere (26). Identity of thisorganism was confirmed by the Center for DiseaseControl, Atlanta, Ga.

Antigens. S. typhi CE was prepared by the modi-fied Schnaitman technique of Hill and Weiss (12) withdiscontinuous sucrose gradients to separate CE fromcytoplasmic membrane, as described earlier (26). Ascoating antigen in the ELISA, 12 lots were prepared,but only one was used to titrate the human sera.Rabbit anti-S. typhi serum (26) was used to determinethe optimal antigen concentration for use in theELISA.

Antigens for slide agglutinations, typhoid H, ty-phoid 0, and paratyphoid A and B, were obtainedfrom commercial sources (BBL Microbiology Systems,Cockeysville, Md.).Human sera. Five sets ofhuman sera were selected

for this study (Fig. 2). The first set comprised 23 singleserum specimens from 11 United States resident lab-oratory personnel and from 12 United States Marinerecruits (the latter kindly furnished by E. Edwards,U.S. Naval Health Science Research Center, SanDiego, Calif.) with no known history of Salmonellainfection or recent vaccination. Sets 2 through 5 werepaired sera collected from hospitalized Indonesian pa-tients who participated in a fever study conducted atthe Jakarta Detachment, U.S. Naval Medical Re-search Unit No. 2 (1). The first serum collected at thehospital was designated acute-phase serum, althoughthe interval between serum collection and day 1 ofrecognized illness was quite variable. The sera col-lected 10 to 14 days later were designated convales-

cent-phase sera. Set 2 was derived from 23 patients forwhom there was no clinical or laboratory evidence ofa Salmonella infection. Set 3 was obtained from 13patients from whom S. typhi serogroup D (12 patients)or S. enteritidis bioserotype paratyphi A (1 patient)was isolated, but agglutination tests were negative(inverse of serum dilution c 20) with serogroup D orA antigens. Two of these patients had titers of 80 withserogroup B antigen. Convalescent sera were obtainedfrom only 5 of these 13 patients. Set 4 was from 26blood culture-negative patients presumed to have hada Salmonella infection on the basis of agglutinationtiters with serogroup D, 0, or H antigens 280. Set 5was derived from Salmonella enteric fever cases di-agnosed by both positive culture and agglutination. S.typhi was isolated from 72 of these patients, and S.enteritidis bioserotype paratyphi A was isolated from9 patients. Included in the definition of agglutinationpositive were all sets of paired sera in which at leastone serum had a titer 240 against serogroup D or Aantigens. However, most of these paired sera had atleast one titer 280 against serogroup D antigens. Ailsera were maintained at -70°C until used.

Microplate ELISA procedure. Ail antigen andserum titrations presented in this communication wereperformed by the ELISA using the alkaline phospha-tase reaction exactly as described by Halle et al. (11)and Dasch et al. (7). Serum IgG and IgM activitieswere titrated by using four to six half-log1o dilutionsteps beginning with 1:160. Both members of a givenserum pair were titrated in the same assay, each assayincluded the same positive and negative referencehuman sera, and all tests were done in duplicate. TheELISA titer was defined as the reciprocal of the serumdilution (obtained from the titration curve by inter-polation) that produced an optical density reading of0.30 (approximately 0.25 optical density unit abovecontrols) at 400 nm after 60 min incubation at 37°C(7).

Ethanethiol treatment. Undiluted human serawere treated with 0.03 M ethanethiol (Eastman Or-ganic Chemical Div., Eastman Kodak Co., Rochester,N.Y.) for 3 h according to a modification of the pro-cedure of Murray et al. (18) described by Halle et al.(11) and used immediately in the bacterial agglutina-tion test employing Salmonella O and H agglutinogens(serogroup D) (Difco Laboratories, Detroit, Mich.).Samples were subsequently incubated uncapped for30 min at 37°C to allow residual reagent to evaporate(11) before use in the ELISA. Also, with each exami-nation of the test specimens, appropriate negative andpositive control sera treated in the same fashion wereincluded as a quality control.

Isoagglutinin determinations. We are indebtedto W. P. Monaghan and N. Oleynik, Blood Bank,National Naval Medical Center, Bethesda, Md., forthe performance of isoagglutinin tests against A and Bblood group antigens, as described by Miller (16).

RESULTSReproducibility ofthe ELISA. The binding

activity of S. typhi CE antigen was tested againsthomologous rabbit antiserum. The results (Fig.1) indicate that 10 ,ug of protein per ml was the

VOL. 13, 1981

on January 3, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

108 BEASLEY, JOSEPH, AND WEISS

2.1 F

1.8

1.5

1.2

o

oIr 0.9o.o

0.6

0.32 1.0 3.2 10 32MICROGRAMS/ML ANTIGEN

FIG. 1. Determination of optimal conceS. typhi CE antigen for coating ofwells in 1The closed circles represent the mean va

CE preparations; the vertical segments i>standard deviations.

smallest concentration that optimallyELISA plates and that variation amonarations was quite small. Subsequeiments with human sera were done witof these preparations which did notserological activity during 20 monthsat -20°C.The human immunoglobulin titers a

CE antigen were also reproducible. Inperformed during a 9-month period, ofpositive and negative reference sera mrat 4°C, the mean titers + standard (

were, respectively, 5,230 ± 670 and 22IgG, and 1,040 ± 230 and invariablyIgM. Comparable titers were obtainedtigen-coated plates were stored for 6 i

-200C before completion of the tests.Titration of human sera by EL

results of a total of 301 assays for IgNantibodies are illustrated in Fig. 2.Of 23 sera (set 1) from individual

nonendemic area with no known histease none had IgM titers >160, wherIgG titers >160 but <500. On the basiresults and the results with the refersthe tentative assumption was madeIgM titers >160 and IgG titers >500cate a significant response to the tesThe validity of this assumption can beby analysis of the other sets in Fig. 2.

Results similar to those described alobtained with 23 paired acute and consera obtained from Indonesian patientnesses unrelated to S. typhi (set 2).IgM titer was >160, and the same ser

moderately high IgG titer (2,300). Ticance of these titers is not known, si

IgM and IgG titers from the convalescent phaseserum from the same patient were <160. An-other convalescent serum IgG titer slightly >500

I was accompanied by an acute-phase serum IgGtiter slightly <500. Most of the other IgG titers>160 but <500 were obtained in both specimensfrom the same patient.

Greater variation in ELISA antibody responsewas encountered in the sera from 13 patientswho were culture positive (S. typhi, 12 isolations;S. enteritidis, 1 isolation) but agglutination neg-ative with serogroup D or A antigens (set 3).Two of these patients (S. typhi isolations) withagglutinins against serogroup B antigen had

100 moderately high acute-phase IgM and IgGELISA antibodies. Unfortunately, the convales-

entration of cent-phase serum from the patient with thethe ELISA. highest acute-phase IgG titer (12,500) was notblues for 12 available. Among the remaining 11 patientsindicate the there were 2 acute-phase sera positive for IgM

(>160) and 5 (including the 2 IgM-positive sera)coated the positive for IgG (>500). The five convalescentg 12 prep- phase sera that were available (including one

nt experi- from the patient with serogroup B agglutinins);h just one had titers comparable to their acute phase coun-

change in terparts. The sera fromn the S. enteritidis patientof storage were negative by ELISA.

Even greater variation was seen in the seragainst the from patients who were culture negative and34 assays, agglutination positive (set 4). Somewhat lessf the same than half of the ELISA IgM titers were positive,maintained and about three-fourths of the IgG titers weredeviations >500. There was an increase in the number of'o ± 55 for IgM-positive reactions in the convalescent sera,<160 for and a greater number of IgG than IgM reactionswhen an- were positive. Otherwise, there was good corre-months at lation between IgG and IgM antibodies and anti-

bodies in the acute- and convalescent-phaseISA. The sera. There was no obvious difference in the4 and IgG agglutination titers of the ELISA-positive and

-negative reactors, with the possible exceptionls from a of slightly greater incidence of agglutination pos-Dry of dis- itive with only H or O antigen in the negativeeas 4 had ELISA reactors.is of these The great majority of acute and convalescentence sera, sera derived from patients who were both cul-that only ture (72 S. typhi and 9 S. enteritidis isolations)may indi- and agglutination positive (set 5) had IgM and,tantigen. IgG antibodies that ranged in titer from 160 toexamined 6,000 and from 360 to 45,000, respectively. The

IgG titers were generally higher (median, 3,500)above were than the IgM titers (median, 600). There was noivalescent obvious difference between titers obtained fromts with ill- acute- and convalescent-phase sera. The latterOnly one observation was confirmed by plotting the acute-um had a phase serum titer against the convalescent-he signifi- phase serum titer of each culture- and aggluti-ince both nation-positive patient (Fig. 3). The results in-

J. CLIN. MICROIBIOL.

r

on January 3, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

SERODIAGNOSIS OF ENTERIC FEVERS BY ELISA 109

SERUM COLLECTIONSET HOSPrML AGGL. CULTURE SPEC NQ

__ SIE 23

2 YES - - ACUTE 23

CONV. 23

3 YES - + ACUTE 13

COus. 5

4 YES + - ACUTE 26

CONV. 26

5 YES + + ACUTE 81

CONV. 81

IgM

o o o o

t e,~ CD O O O O O ao

e_* * .e.. *.. . .~8Soe§88588 888§}o§ og% o8 o8 8j * M.ds4.ei- -M4 3.4 S a m*Oe

iNO - - SPEC.23 000 0 OgG

2 YES - - ACUTE 23 oo60 0 0 0

CONV 23 a* e *g e

3 YES - + ACUTE 13 § 0 O 0 Oo 0o 0

CONV. 5 * * * * e

4 YES + - ACUTE 26 8 o o o o o o oo oo 8 oo oo o o oo

CONV. 26 * * * * * a e & 8 * *0

5 YES + + ACUTE 81 O 00o 8o 88 à8fo 8 808 8 oCONV. 81 eg m a a

S 160 500 1600 1>00 16,000 50,000

ELISA TITER (INVERSE OF SERUM DILUTION)

FIG. 2. Distribution ofIgM and IgG antibodies against S. typhi CE antigen for five sets ofhuman sera, asdescribed in the text and summarized here. HOSPITAL., hospitalizedpatients; AGGL., agglutination; SPEC.,specimen, acute- or conv.- (convalescent) phase sera. (O) Single or acute-phase sera; (S) convalescent-phasesera.

dicate that, although there is considerable indi-vidual variation, the correlation (r = 0.80) be-tween IgM or IgG titers in the acute- and con-valescent-phase sera is good. There is no indi-cation of a trend towards higher or lower titersin the convalescent-phase sera. To test the spec-ificity of the reaction, a few sera in this set weretested in an ELISA employing Escherichia coliK-12 CE as the coating antigen. These sera wereuniformly nonreactive against the E. coli CE.Moreover, there was no reduction in ELISA IgMtiters after the absorption ofrepresentative IgM-positive sera with an excess of heat-aggregatedhuman IgG bound to Sepharose 4B, indicatingthat the IgM titers were not influenced by arheumatoid factor.

In Fig. 2, the 84 paired sera derived frompatients from whom S. typhi was isolated werenot separated from the 10 paired sera associatedwith S. enteritidis infection, since the aggluti-nation titers with serogroup D antigen weregenerally higher than with serogroup A antigen.The ELISA titers of the 10 paired sera from theS. enteritidis patients are illustrated in Fig. 4.

The patterns are very similar to those of theother sera, except that the median titers aresomewhat lower (400 and 1,500 for IgM and IgG,respectively) even when the sera from the agglu-tination- and ELISA-negative patient are ex-cluded. Because of the high degree of variationin this small sample, the significance of thisreduced reactivity is not apparent.Ethanethiol treatment of the sera. Eth-

anethiol has been used to selectively destroyIgM to demonstrate the presence of this immu-noglobulin class in immune sera (18), and thistreatment has been shown to be compatible withthe ELISA procedure (11). Serum specimensfrom seven proven Salmonella infections weretreated with the reagent, and residual antibodyactivities were then measured with both theELISA and agglutination tests. Effects of eth-anethiol on the serological activities of thesesera are shown in Table 1. The ELISA IgG titerswere not appreciably affected by this treatment,but IgM titers were drastically reduced. Theconcomitant reductions in O and H agglutininactivities confirm the finding (5, 6, 13) that the

VOL. 13, 1981

0

on January 3, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

110 BEASLEY, JOSEPH, AND WEISS

w

QizwhJ(nw

-J

w

Ig M

* 0r 80

Ig G

r= 80

<22 25 30 35 30 35 40 45

LOG ELISA TITERS (ACUTE SERA)

FIG. 3. Correlation between acute- and convalescent-phase serum titers, IgM, and IgG as determined byELISA from culture- and agglutination-positive patients. The solid lines are the linear regressions of theexperimental log ELISA titers of acute- and convalescent-phase sera; r is the correlation coefficient. Dottedlines represent locus ofpoints, log ELISA acute serum titers = log ELISA convalescent serum titers.

IgMACUTE

CONV.

IgGACUTE

CONV.

Ce O OCD O O

*% .a

O o o o 8 c0

a 160 500 1600 5000

ELISA TITER (INVERSE OF SERUM DILUTION)FIG. 4. Distribution of IgM and IgG antibodies

against S. typhi CE antigen from 10 patients fromwhom S. enteritidis, bioserotype paratyphi A, wasisolated. (O) Acute-phase sera; (O) convalescent-phase sera.

agglutination reaction is brought about primar-ily by IgM antibodies.Correlation between ELISA and aggluti-

nation titers. The sera from set 5 of confirmedSalmonella patients were divided into subsetsof identical H or O antigen agglutination titers.The geometric means ± standard deviations ofthe ELISA IgM titers of these subsets of sera

are shown in Fig. 5. There is some correlationbetween ELISA IgM and O agglutination titers,but not necessarily H agglutination titers, in theacute-phase sera (A and B). No correlation canbe detected in convalescent-phase sera (C andD) or between H or O agglutination and ELISAIgG titers (data not shown).Blood group distribution of patients in

relation to the antibody response. Intestinalbacteria have been implicated in the stimulation

TABLE 1. Effect of ethanethiol treatment onantibody titers of sera from selected patients

ELISA titer Agglutinin ti-ter<Patient Serum treat-Ptet ment O H

IgG IgM Anti- Anti-gen gen

1 None 4,200 3,800 320 20Ethanethiol 4,500 600 40 <20

2 None 12,500 1,300 320 320Ethanethiol 12,000 125 <20 <20

3 None 7,000 260 <20 160Ethanethiol 6,000 <50 <20 20

4 None 6,000 270 <20 80Ethanethiol 6,500 <50 <20 <20

5 None 5,500 2,700 320 320Ethanethiol 5,000 160 <20 40

6 None 4,200 270 40 320Ethanethiol 4,300 <50 <20 80

7 None 1,600 900 80 <20Ethanethiol 1,500 50 <20 <20

Comparable results were obtained with ELISAIgM-negative sera.

of isoagglutinins, but this stimulation is limitedby the presence of the corresponding bloodgroup antigens (27). It is reasonable to expect,therefore, that the antibody responses oftyphoidand paratyphoid patients might be influenced insome way by their blood group. This possibilitywas investigated by determining the presence orabsence of anti-A and anti-B isoagglutinins (andthus, indirectly, the blood group) of 118 patientswith confirmed or presumptive diagnosis of ty-

J. CLIN. MICROBIOL.

'

on January 3, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

SERODIAGNOSIS -OF ENTERIC FEVERS BY ELISA 111

w

wQ z

oL

3

A

>l-

B

cC D

3 >-+ -

2_____ __ ___ __ _ __ __ _ ___~~~~

S 20 40 80 160 O320 S20 40 80 160 ?320H ANTIGEN 0 ANTIGEN

AGGLUTINATION TITERS

FIG. 5. Correlation between agglutination andIgM ELISA titers. The closed circles represent thegeometric mean ELISA titers; the vertical segmentsindicate the standard deviations. (A and B) acute-phase sera; (C and D) convalescent phase sera; (Aand C) H agglutination; (B and D) O agglutination.

TABLE 2. Antibody response as determined byagglutination in confirmed andpresumptive casesoftyphoid andparatyphoid lever in relation to

blood groupAgglutination' Blood groupb

(antigen) oaSet Cultureb otalten

D and/ B p A B ABorA only OABA

3a + - - 11 1 6 3 13b + - + 2 1 14 - + - 25e 17 5 2 15 + + - 80e 36 21 19 4

a Determined by the presence of isoagglutinins inserum.

b Isolation of S. typhi or S. enteritidis.c As defined in Materials and Methods.d Probability of random distribution by chi square

<5% in the following comparisons: blood groups A/Oset 3a versus set 4 or 5; A + B + AB/O set 3a versusset 4 or sets 3b + 4 + 5.

e One patient omitted because of insufficient serum.

phoid or paratyphoid fever (from sets 3, 4, and5). The results are shown in Tables 2 and 3.Of particular interest is the group of 11 pa-

tients (Table 2, set 3a) from whom S. typhi or S.enteritidis had been isolated without the dem-onstration of the corresponding agglutinins oragglutinins against serogroup B. Of these pa-tients only one was blood group 0. In compari-son with blood group A (6 patients) or all theother 10 patients, the distribution is significantlydifferent from that of patients who developedagglutinins (Table 2, sets 3b, 4, and 5). If theseresults can be confirmed, they would mean thatblood group O patients are the most likely todevelop antibodies in response to Salmonella

TABLE 3. Antibody response as determined byELISA in confirmed andpresumptive cases of

typhoid andparatyphoid lever in relationship toblood group

Bacterium iso- ELISAb Total Blood group

lated patients 0 A B ABS. typhi IgM- 12 3 6 2 1

IgM+ 71 31 20 16 4IgG- 5 3 1 1IgG+ 78 34 23 17 4

S. enteritidis IgM- 3 2 1IgM+ 7 3 2 2IgG- 2 1 1IgG+ 8 4 2 2

Nonec IgM- 13 10 2 1IgM+ 12 7 3 1 1IgG- 5 4 1IgG+ 20 13 4 2 1

a Determined by the presence of isoagglutinins inserum.

b IgM+ is defined as serum titer >160; IgG+ isdefined as a titer of >500 in at least one of two sera.

C Agglutination positive.

antigens. There is a slight preponderance ofblood group O patients among those who wereagglutination positive but culture negative (Ta-ble 2, set 4), but the difference in distributionbetween this series and the patients who wereboth culture and agglutination positive (set 5) isnot significant.Table 3 illustrates the division of the sera from

the same 118 patients on the basis of the ELISAresults. Although most of the 11 patients in set3a (Table 2) were IgM negative by ELISA, a fewsera from the other sets were IgM negative aswell. The net result is a blood group distributionamong IgM-negative patients that does not dif-fer significantly from that of the majority. Thesame is true of the distribution among IgG-neg-ative patients, possibly because their numberwas small (Table 3, S. typhi isolations) or Sal-monella infection had not been confirmed (Ta-ble 3, no bacterium isolated).

DISCUSSIONThe ELISA, as performed in this study, meets

the requirements of objectivity, reproducibility,sensitivity, and simplicity. Some of the earlierdifficulties encountered by Sippel et al. (26),resulting in a relatively low span of difference inoptical density readings between positive andnegative sera, were eliminated by strict adher-ence to the protocols of Halle et al. (11) andDasch et al. (7) (Fig. 1). The amount of antigenrequired for the test (10 ,ug/ml) is comparable to

VOL. 13, 1981

on January 3, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

112 BEASLEY, JOSEPH, AND WEISS

that used by the investigators cited above andby others (3, 4, 29). It was shown by Halle et al.(11) that the reaction is improved by the removalof extraneous components from the antigenpreparation. We selected, however, a crude an-tigen, containing O antigen 12, common to threeSalmonella serotypes frequently pathogenic inhumans, which could be expected to be broadlyreactive at the expense, perhaps, of the serolog-ical specificity achieved by Svenungsson et al.(29). The results shown in Fig. 2 are endpointtitrations, but, as in the case of other studies (7,11, 29), entirely satisfactory results can be ob-tained within limits with appropriate single se-rum dilutions, thereby permitting an estimationof titers directly from optical density values (notshown). Although a spectrophotometer is con-venient, under field conditions a semiquantita-tive estimation of serum titers can be obtainedby visual inspection (7, 11). The usefulness ofthe precoated plate, demonstrated in this study,may further contribute to the simplicity andreproducibility of the test under field conditions.The sera used in this study come from a

variety of clinical situations. If the criterion isadopted that IgM titers <160 and IgG titersc500 are negative, all sera from individuals froma nonendemic area with no known history ofdisease were negative. These results comparefavorably with those of Carlsson et al. (4), whoobtained somewhat higher titers against sero-group D and B antigens in his control sera. Asexpected, in a comparable examination of con-trol sera from an endemic area, an occasionalserum was positive. It is not known whether thiswas due to previous exposure to Salmonellaantigens or to unrelated causes. When the diag-nosis of typhoid or paratyphoid fevers was pre-sumptive, i.e., when either the agglutination testor blood culture was negative, the ELISA con-firmed the presence of IgM or IgG antibodies insome patients, but five patients in each groupremained negative (Table 3). It appears, there-fore, that additional blood cultures (only oneattempt was made per patient) or more extensiveserology would have narrowed the number ofunconfirmed Salmonella diagnoses, but somestill would have remained unconfirmed. Theseresults contrast with the confirmation of anti-bodies by ELISA in that at least one serunspecimen in all culture- and agglutination-posi-tive patients was positive. The value of deter-mining IgG as well as IgM antibodies with theELISA, as previously illustrated by Svennugs-son et al. (29), is obvious, since both the fre-quency of occurrence and titer of IgG antibodieswere higher in our study.The well-established criterion for serological

diagnosis by rising antibody titers in paired serawas not valid in this study. It was not possibleto extract from the data shown in Fig. 2 even asmall but significant subset of paired sera inwhich a clear rise in titers did occur. This findingcan be attributed to several causes. Most of thepatients who sought admission to the hospitalhad been febrile for several days and might haveachieved peak antibody titers at the time thefirst blood specimen was withdrawn. Some pa-tients might have been antigenically stimulatedthrough inapparent infection before illness andmight have responded anamnestically. In addi-tion to serotype D, serotype A strains are appar-ently quite common in Indonesia (22, 23), andserotype B strains have been isolated from sur-face waters in Jakarta (10). There is some indi-cation that anamnestic responses have occurredin some of the patients of this study, because, atleast in the agglutination test, the patients didnot always respond preferentially to the sero-group antigen of the Salmonella species thatwas isolated from them. This aspect was notstudied in the ELISA, but it is obvious (Fig. 4)that the patients reacted reasonably well withheterologous antigens. The effect of chloram-phenicol treatment on the serological responseis not known, but the ELISA titers of 32 patientswho claimed to have received chloramphenicolprior to hospital admission were not differentfrom those of 28 patients who claimed not tohave been so treated (data not shown).The chemical composition of the CE antigen

used in the ELISA most closely resembles the Oantigen of the agglutination test. It is not sur-prising, therefore, that there was some correla-tion between the IgM response demonstrated byELISA and O agglutination in the acute-phasesera (Fig. 5), and in this respect the results agreewith those of Carlsson et al. (4). However, othercorrelations in titer could not be made, eventhough the overall correlation between positiveELISA and positive agglutination is good (Fig.2). These results indicate that the ELISA, evenwith a crude antigen, measures antigen-antibodyreactions not detected by agglutination (Table1) which is heavily biased for IgM detection. Inour limited experience, an Enterobacteriaceaecommon antigen or rheumatoid factor did notinfluence the ELISA reaction.The lack of agglutinin development in some

patients with well-documented typhoid fever,including human volunteers, has surprised anumber of investigators (14, 25, 28). The resultsshown in Table 2 which suggest some correlationbetween blood group and diminished agglutininresponse against Salmonella antigens, not nec-essarily reflected in the ELISA (Table 3), should

J. CIAIN. MICROBIOL.

on January 3, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

SERODIAGNOSIS OF ENTERIC FEVERS BY ELISA 113

be viewed with caution, since a relatively smallnumber of patients was involved and the sero-logical studies have not been extensive. How-ever, this observation cannot be disregardedsince a somewhat similar association involvingblood group B individuals has been suggestedfor the Weil-Felix Proteus OX-19 reaction fortyphus fevers (30). If such investigations arepursued or extended to reported instances ofassociation between certain human blood groupor leucocyte antigens and some types of Salmo-nella infections (reviewed in 15, 17), the ELISAwould be the technique of choice because of itsobjectivity and versatility.

In conclusion, this study has provided goodevidence that the ELISA performed with a sin-gle CE antigen derived from S. typhi is a valuableaid to the diagnosis of Salmonella infection inan endemic area and is a suitable alternative tothe agglutination test as it is performed in manylaboratories.

ACKNOWLEDGMENTSWe thank Holly R. Labore for her excellent technical

assistance, Sidney Halle for furnishing standardized reagents,and Herbert R. Morgan for valuable discussions. We alsothank Donna Boyle for the expert secretarial preparation.Supply of rheumatoid factor absorbent by Richard Wistar isespecially appreciated.

This work was supported by the Naval Medical Researchand Development Command, Research Work Unit no.MR0410501.0044.

LITERATURE CITED

1. Anderson, K. E., S. W. Joseph, R. Nasution, Sunoto,T. Butler, P. F. D. Van Peenen, G. S. Irving, J.Sulianti Saroso, and R. H. Watten. 1976. Febrileillnesses resulting in hospital admission: a bacteriologi-cal and serological study in Jakarta, Indonesia. Am. J.Trop. Med. Hyg. 25:116-121.

2. Benedict, A. A. 1965. Sensitivity of passive haemagglu-tination for assay of 7S and 19S antibodies in primaryrabbit anti-bovine serum albumin sera. Nature (Lon-don) 206:1368-1369.

3. Carlsson, H. E., A. A. Lindberg, and S. Hammar-strom. 1972. Titration of antibodies to Salmonella Oantigens by enzyme-linked immunosorbent assay. In-fect. Immun. 6:703-708.

4. Carlsson, H. E., A. A. Lindberg, S. Hammarstrom,and A. Ljunggren. 1975. Quantitation of SalmonellaO antibodies in human sera by enzyme-linked immu-nosorbent assay (ELISA). Int. Arch. Allergy Appl. Im-munol. 48:485-494.

5. Chau, P. Y., and M. H. Ng. 1978. Differential aggluti-nation of particulate Vi and O antigens by the IgM andIgG class antibodies. Aust. J. Exp. Biol. Med. Sci. 56:39-45.

6. Chernokhvostova, E., K. I. Luxemburg, V. Starshi-nova, N. Andreeva, and G. German. 1969. Study onthe production of IgG-, IgA-, and IgM-antibodies tosomatic antigens of Salmonella typhi in humans. Clin.Exp. Immunol. 4:407-421.

7. Dasch, G. A., S. Halle, and A. L. Bourgeois. 1979.Sensitive microplate enzyme-linked immunosorbent as-

say for detection of antibodies against the scrub typhusrickettsia, Rickettsia tsutsugamushi. J. Clin. Microbiol.9:38-48.

8. DeVillier, A. B., R. H. Deupree, C. Dickinson, and M.F. Beeler. 1965. Comparative study of typhoid O anti-gens. Am. J. Clin. Pathol. 44:410-412.

9. Gardner, A. D. 1937. An international experiment on theWidal-reaction. J. Hyg. 37:124-142.

10. Gracey, M., P. Ostergaard, S. W. Adnan, and J. B.Iveson. 1979. Faecal pollution of surface waters inJakarta. Trans. R. Soc. Trop. Med. Hyg. 73:306-308.

11. Halle, S., G. A. Dasch, and E. Weiss. 1977. Sensitiveenzyme-linked immunosorbent assay for detection ofantibodies against typhus rickettsiae, Rickettsia prow-azekii and Rickettsia typhi. J. Clin. Microbiol. 6:101-110.

12. Hill, J. C., and E. Weiss. 1974. Protein fraction withimmunogenic potential and low toxicity isolated fromthe cell wall of Neisseria meningitidis group B. Infect.Immun. 10:605-615.

13. Kumar, K., A. N. Malaviya, R. G. S. Murthy, M.Venkataraman, and L. N. Mohapatra. 1974. Immu-nological study of typhoid: immunoglobulins, C3, anti-bodies, and leukocyte migration inhibition in patientswith typhoid fever and TAB-vaccinated individuals.Infect. Immun. 10:1219-1225.

14. Levine, M. M., 0. Grados, R. H. Gilman, W. E. Wood-ward, R. Solis-Plaza, and W. Waldman. 1978. Di-agnostic value of the Widal test in areas endemic fortyphoid fever. Am. J. Trop. Med. Hyg. 27:795-800.

15. McMichael, A., and H. McDevitt. 1977. The associationbetween the HLA system and disease, p. 39-100. In A.G. Steinberg, A. G. Bearn, A. G. Motulsky, and B.Childs (ed.), Progress in medical genetics, vol 2. W. B.Saunders Co., Philadelphia.

16. Miller, W. 1977. Technical manual, p. 89-90. AmericanAssociation of Blood Banks, Washington, D.C.

17. Mourant, A. E., A. C. Kopec, and K. Domaniewska-Sobczak. 1978. Blood groups and diseases: A study ofassociations of diseases with blood groups and otherpolymorphism. Oxford University Press, New York.

18. Murray, E. S., J. M. O'Connor, and J. A. Gaon. 1965.Differentiation of 19S and 7S complement fixing anti-bodies in primary versus recrudescent typhus by eitherethanethiol or heat. Proc. Soc. Exp. Biol. Med. 119:291-297.

19. Neter, E. 1956. Bacterial hemagglutination and hemoly-sis. Bacteriol. Rev. 20:166-188.

20. Protell, R. L., R. D. Soloway, W. J. Martin, L. J.Schoenfield, and W. H. J. Summerskill. 1971. Anti-salmonella agglutinins in chronic active liver disease.Lancet ii:330-332.

21. Reynolds, D. W., R. L. Carpenter, and W. H. Simon.1970. Diagnostic specificity of Widal's reaction for ty-phoid fever. J. Am. Med. Assoc. 214:2192-2193.

22. Sanborn, W. R., R. Hablas, S. Komalarini, Sinta, R.Trenggonowati, T. Sadjimin, Atas, and Sutrisna.1977. Salmonellosis in Indonesia: phage type distribu-tion of Salmonella paratyphi A. J. Hyg. 79:1-4.

23. Sanborn, W. R., J. F. Vieu, S. Komalarini, Sinta, R.Trenggonowati, I. L. Kadirman, Sumarmo, A.Aziz, T. Sadjimin, Triwibowo, Atas, and S. Ayam.1979. Salmonellosis in Indonesia: phage type distribu-tion of Salmonella typhi. J. Hyg. 82:143-153.

24. Sansone, P., M. S. Saslaw, and C. H. Hennekens, Jr.1972. High titer Widal reaction. J. Am. Med. Assoc.220:1615-1616.

25. Schroeder, S. A. 1968. Interpretation of serologic tests

VOL. 13, 1981

on January 3, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

114 BEASLEY, JOSEPH, AND WEISS

for typhoid fever. J. Am. Med. Assoc. 206:839-840.26. Sippel, J. E., H. K. Mamay, E. Weiss, S. W. Joseph,

and W. J. Beasley. 1978. Outer membrane proteinantigens in an enzyme-linked immunosorbent assay forSalmonella enteric fever and meningococcal meningitis.J. Clin. Microbiol. 7:372-378.

27. Springer, G. G., and R. E. Horton. 1969. Blood groupisoantibody stimulation in man by feeding blood group-active bacteria. J. Clin. Invest. 48:1280-1291.

28. Stuart, B. M., and R. L. Pullen. 1946. Typhoid: clinicalanalysis of three hundred and sixty cases. Arch. Intern.Med. 78:629-661.

29. Svenungsson, B., H. Jorbeck, and A. A. Lindberg.1979. Diagnosis of salmonella infections: specificity of

J. CLIN. MICROBIOL.

indirect immunofluorescence for rapid identification ofSalmonella enteritidis and usefulness of enzyme-linkedimmunosorbent assay. J. Infect. Dis. 140:927-936.

30. Tamiya, T., H. Hazato, T. Yamamoto, T. Iida, H.Shimojo, K. Nishioka, A. Kawamura, K. Suzuki,M. Arai, R. Tsukamoto, and Y. Schoble. 1949. Stud-ies on the so-called X-factor common to Proteus OX19,Rickettsia prowazeki and Rickettsia mooseri, espe-cially on the relation of the factor to blood group Bspecific substance. Jpn. J. Exp. Med. 20:1-23.

31. Zinner, S. H., and W. R. McCabe. 1976. Effects of IgMand IgG antibody in patients with bacteremia due togram-negative bacilli. J. Infect. Dis. 133:37-45.

on January 3, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from