Diagnostic implications of parasite specific immune responses in immunocompromised patients with strongyloidiasis

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0095-1137/86/061099-05$02.00/0

Diagnostic

Implications of Parasite-Specific Immune

Responses

in

Immunocompromised

Patients with Strongyloidiasis

ROBERT

M.

GENTA,1*

RICHARDW. DOUCE,2AND PETER D. WALZER2

Department ofPathology and Laboratory Medicine' andDepartment of Medicine, Division ofinfectiousDiseases,2 VeteransAdministration Hospital and University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0529

Received24January 1986/Accepted4March1986

Parasite-specifichumoral and cellular immuneresponseswereevaluatedin nine immunosuppressedpatients

withStrongyloides stercoralis infection. Four patients had the disseminated form ofthedisease, and five had serious manifestations without evidence of hyperinfection. All patients had highlevelsofimmunoglobulin G (IgG) antibodies directed against S. stercoralis larval antigensasdetectedbytheenzyme-linked immunosorbent

assay. Seven patients had both elevated totalserum IgEand parasite-specific IgE antibodiesdetected by a

modified radioimmunoassay. The hyperinfected patients differed significantly fromthenonhyperinfectedgroup

hi totaland specific IgE levelsandperipheral eosinophilia butnotinspecific IgGlevels. Noneof the patients

testedexhibited significant in vitro lymphoproliferative responsestoparasite antigens. Our results indicated that the possibility ofaprotective role of IgE and eosinophils in strongyloidiasis deserves further investigation.

Inaddition, wesuggestthat theenzyme-linked immunosorbentassayforspecific IgG antibodiesmayhavean

important place in thediagnosis of strongyloidiasis in immunocompetent aswellasinimmunocompromised

patients.

Strongyloides stercoralis is an intestinal nematode of

worldwide distribution. The unusual autoinfectious cycle of this parasite allows its persistence in the infected host for indefinite periods, often as a well regulated, undetected

parasitosis(13, 15). When the infectionescapeshost control, as happens in immunosuppressed individuals harboring the

parasite, it may become a disseminated, life-threatening

disease (31). Thus, S. stercoralis has emergedas an

impor-tant cause of morbidity and mortality in immunocompro-mised patients (20).

Theparasitologicaldiagnosisofstrongyloidiasis maybea

difficult task. Because of the irregular larval output by the intestinal adult females, the diagnostic sensitivity of stool examination is low (29). Other methods such as fecal

cul-tures, baermanization, or gastrointestinal aspiration have

been reported to be more sensitive (21, 30), but they are

usually beyond the scope of most clinical microbiology laboratories. Recently, an enzyme-linked immunosorbent

assay (ELISA) (2, 28) and anindirect immunofluorescence

test (12, 17) have been reported as being sensitive and specific adjuncts for the diagnosis of strongyloidiasis. In addition, we have developed a radioallergosorbent test

(RAST)todetectimmunoglobulinE(IgE) antibodies against S.stercoralislarvalantigens. Theseimmunoserologicaltests

have been evaluated for series of patients with chronic, uncomplicated strongyloidiasis andapresumably intact

im-mune system. Arapid diagnosis, however, iscrucial in the

presence ofdisseminated disease, which frequently occurs

in patients who have been receiving immunosuppressive therapy, particularly corticosteroids (6).

Over the past few years we had the opportunity of

studying nine immunosuppressed patients with serious S. stercoralis infection. Our results indicate that specific antilarval IgG and IgE antibodies persist even after

pro-longed steroid therapy and that they can be usefully

ex-ploited forimmunodiagnosis.

*Correspondingauthor.

MATERIALS AND METHODS

Patients. All nine patients had S. stercoralis infection documented by parasitological examination of stools, duodenalaspirate,orboth. Infour subjects (A,B,C, and D),

larvae were also identified in the sputum. The patients' relevantclinical featuresaresummarized inTable 1. Patient

Ahas beenpreviously reported elsewhere (24).

Immunological studies. (i) ELISA. A modified version of the ELISA test described by Neva et al. (28) was used.

Briefly, S. stercoralis soluble (SS) antigens wereextracted

fromfilariform larvae obtainedfrom fecal cultures of

exper-imentally infected dogs maintained as larval donors (11).

Alternate rows of polyvinyl microtiter plates (Dynatech

Laboratories, Inc., Alexandria, Va.) were sensitized with

theantigen. Eachserum wastestedatasingle dilution of 1:8

both inawell withantigen and inawell withoutantigen. The

ELISAwascompleted byusinggoatanti-humanIgG conju-gated with alkaline phosphatase (Miles-YedaLtd., Rehovot, Israel) and p-nitrophenylphosphate disodium salt as the

substrate. The net absorbancy of each test serum was

obtainedbysubtractingthe absorbancy ofthe well without antigen from that of the sensitized well. The results are

reported as the Absorbancy Index (AI), which was calcu-latedby dividingthenetabsorbancyof thetestserumbythe

net absorbancy ofa high-positive control present on each

plate. This value islinearlyrelatedtotheantibodytiters. In standardization experiments, 99 of 100 presumably noninfected, healthyNorth American blood donors had AI values below0.15, andonehadvalues above 0.15 but below 0.30; 49 of 50 patients with parasitologically proven S. stercoralis infection and no history ofimmunosuppression hadAI values higherthan0.30, and onehad avalueabove 0.15 but less than 0.30. Thus, an AI less than 0.15 was

regarded as negative, a value between 0.15 and 0.30 was

regarded as borderline, and a value greater than 0.30 was

regarded as positive. When positive sera from infected

patients were preabsorbed with SS antigens, the AIvalues

consistently became negative (<0.15). Preabsorption with 1099

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TABLE 1. Patient characteristics and clinical features

Patient Geographic Underlyingillness(immunosuppressivetreatment) Clinicalfeaturesofstrongyloidiasis

(age/sex) history

A(59/M) Cuba (in U.S. Systemic lupuserythematosus (prednisone,80 mg Hyperinfection (gram-negative meningitis, cerebral 18 yr) qd altwith30mgqd x 7yr) vasculitis, abdominal pain, larvae in stools and

sputum)

B(23/F) Nicaragua(in Systemic lupus erythematosus Hyperinfection (E. colisepsis, meningitis, U.S.6yr) (methylprednisolone, 40mgqd x 8mo) abdominalpain, larvae instools)

C(69/M) Kentucky, Idiopathicthrombocytopenicpurpura Hyperinfection (Pseudomonas meningitis,adult SouthPacific (prednisone, 40 mg qd x 4 mo) respiratory distress syndrome, larvae in stools

andsputum)

D(53/M) Kentucky Chronicobstructive lungdisease,carcinoma of Hyperinfection (hemoptysis, respiratory

prostate(methylprednisolone, [?] dose x 18 insufficiency, pneumonia, diarrhea, disseminated weeks) aspergillosis [late complication], larvae in stools

andsputum)

E(55/F) Kentucky Rheumatoidarthritis, S. aureuscellulitis Diarrhea,larvaein stools (prednisone, 5 mgqd + azathioprine, 50 mgqd

x [?] duration)

F(63/F) Kentucky Autoimmunehemolyticanemia Bloodydiarrhea, vomiting, coloniculcerations, (cyclophosphamide, 50mgqd + indomethacin, larvae in duodenalaspirate

75mgqd x 8mo)

G(44/M) Kentucky Diffuseproliferativeglomerulonephritis Postprandialnausea, duodenalulceration,larvae (prednisone, taperingdoseto20 mgqd x 2mo in stools

+ nitrogen mustard, 35 mg/kgqd x 3wk)

H(83/M) Kentucky Redcellaplasia (prednisone,60mgqd x 4mo) Asthma(questionably related), larvae in stools I(63/M) Ohio Chronic activehepatitis(prednisolone, 10-25 mg Severediarrhea,wtloss

qd x 2yr)

Ascaris suumantigens reducedthe AIless than

8%,

whereas

Ancylostoma

caninumdidnotaltertheabsorbance. (H) RAST. The

Phadebas-RAST

radioimmunoassay (Pharmacia Diagnostics, Piscataway, N.J.) was

adapted

for thedetection ofantistrongyloides IgE. Disks (20mg; 7 mm)

were punched out of#595 filter paper and activated with

cyanogen bromide as described

by

Lee and Heiner

(24).

Activated disks were then incubated with S. stercoralis

larval antigen, preparedasdescribedabove,at a

concentra-tion of 100

pgg/ml

for 18 hat4°C and stored in RAST buffer (Pharmacia) untilused.

Samples

(100 ,ul) ofeach test serum were incubated overnight at room temperature with an

activated disk, washed three

times

in RAST

washing

solu-tion (Pharmacia), and incubated overnight at room temper-ature with 50

pil

of

'25I-labeled

anti-IgE. After three more

washings, the radioactivity present on each disk was

countedin a gamma counter(PackardInstrumentCo.,Inc.,

Rockville, Md.). Eachserum wastested induplicate,and the mean of the counts was usedfor calculations. Results are

expressed

aspercent

binding, which

is theratio between the countsper minuteofeach test disk and the

total

radioactivity

present in a sample of

50

,ug of

125I-labeled

anti-IgE. This value is linearly related to the amount of specific IgE present in the serum (24).

In standardization experiments, 47 of 49 presumably

noninfected, healthy North Americans blood donors had

bindingvalueslowerthan1.1%and two had valuesbetween

1.1% and

3.2%.

Of50 nonimmunosuppressed patients with

parasitologically proven S. stercoralis infection, 48 had

bindingvalueshigher than 3.2% and two had values between

1.1% and 3.1%. We therefore regarded values higher than

3.2% as positive. Values below 1.1% were regarded as

negative,andvaluesbetween 1.1% and3.2%were

regarded

asborderline.

Initial experiments suggested that

using

undiluted sera

provided the best discrimination between infected and noninfected

patients.

The useofundilutedserummay have

accounted for the

high binding

values found in

nonhyperinfected patients. Preabsorption of patients' sera

with A. suum or A. caninum antigens did not alter their

binding values,

whereas

preabsorption

with SS

antigens

caused an average 83% decrease oftheirbindingvalues. (iii) Total IgE determination. Total serum IgE was mea-sured with a standard radioimmunosorbent technique (PRIST, Pharmacia).Results wereexpressed in international units. Values below 250 IU are considered normal at the

University of Cincinnati Diagnostic Immunology

Labora-tory.

(iv) Lymphocyte

proliferation.

Invitrolymphoproliferative

responses ofperipheral lymphocytes to SS antigens were

assayedaspreviously reported (9) for four patients. Results areexpressedasthelymphoproliferative index (E/C),which is the ratio between responses ofantigen-stimulatedcultures (E) and those of unstimulated cultures (C). The values

reported represent each patient's maximal response to

var-ious concentrations of SSantigens (between 1 and 20 ,ug of

protein per ml). Maximal responses to the mitogen

phytohemnagglutinin

arealso reportedforcomparison.

RESULTS

Clinical features. Relevant clinical data of the study pa-tients are

depicted

in Table 1. Six weremales and three were

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TABLE 2. Immunologicalcharacteristics of patients

Range of no. of Lymphoproliferative

Patient

eyhofytes

of TotalIgE SpecificIgE Anti-SSIgG response(EIC)a

Patient erythrocytes(%

(lU)

(%binding) (AI)

eosinophils) PHA SS-Ag

A 600-14,000 (0-0) 260 4.8 Q.420 30 2.3

B 7,600-8,600 (1-2) 380 22.8 0.737 ND ND

C 19,000-9,800 (0-2) 30 2.0 0.830 ND ND

0.450b

D 9,800 (5) 32 2.3 1.275 ND ND

E 5,600-6,700 (9-20) 1,050 30.3 0.940 41.3 1.5

F 11,000-11,200 (19-43) 380 31.6 1.180 12.3 2.0

G 9,900-11,000 (4-8) 3,000 24.3 1.402 ND ND

H 9,000-13,000 (7-24) 5,000 37.3 0.950 6.5 3.9

I 3,800-5,600 (3-13) 6,200 39.5 1.230 ND ND

aLymphoproliferative responses to phytohemagglutinin (PHA) and to S.stercoralisantigens (SS-Ag) are expressed as theratio between stimulated(E)and

unstimulated (C) cultures. ND, Not done.

bForpatientC,thesecond value indicates the AI of the cerebrospinal fluid.

females, andthe age range was 23 to 83. Three patients had ahistory ofoverseastravelto areasin whichthe diseasewas

endemic; six patients were or had been residents of rural

Kentucky, an area shown to have a high prevalence of

strongyloidiasis (33). All patients had underlying diseases that were

being

treated with corticosteroids for periods varying from several

months

to over 10 years. For some

patients other immunosuppressive drugs were also being

used.

According to the clinical manifestations of

stron-gyloidiasis, the patients could be divided into two groups.

Patients A, B, C, and D had the classical features of S. stercoralis hyperinfection, with pneumonia, sepsis, or

men-ingitis and large numbers oflarvae present in the stools, sputum, or both. Two of these patients (C and D) died

despite thiabendazole therapy.The other twopatientsin this groupreceived multiplecourses ofthiabendazoleand

recov-ered, with disappearance ofthe parasites from the stools at

follow-up examinations.

Theonly manifestation of strongyloidiasis in patients E, F,

G,

and I were gastrointestinal symptoms; patient H had asthma thatwas questionably related

to

theparasite. All of thesepatients had larvae in their stools, butnonehadsigns

or

symptoms

suggestive ofhyperinfection. Thiabendazole

treatment cleared the infection and resolved the symptoms in allthesepatients.

Immunology. The patients' relevant laboratory data and theresultsof theimmunologictests aredisplayed in Table2.

Forseveralpatients,multipleserumsampleswereavailable;

the data shown represent values obtained from the serum

drawnatthe time ofdiagnosis.

Allpatients had high titers of specific IgG,asindicatedby theirAI values. The mean AIs ± standarddeviations were

0.872 ± 0.455in the

hyperinfected

groupand1.140 ± 0.196 in the group without

hyperinfection.

When compared by using the Wilcoxon rank sum test, the difference between

groups was notstatistically significant.

TotalIgEwaselevated inseven

of

the nine

patients

(77%).

The mean values + standarddeviationswere 175.5± 173in the hyperinfected group and 3,126 ± 2,491 in the

nonhyperinfected group.The difference betweenthegroups was significant(P < 0.05).

Specific IgE values, expressedas mean ± standard devi-ation of the percent binding, were also lower in the

hyperinfected (7.9 ± 9.9)than in the

nonhyperinfected (32.6

± 6.0) patients, but the difference between them was not

statistically

significant.

Several eosinophilic counts were available for each

pa-tient, andconsiderable day-to-day variation was present in mostof them. However, when the meanoftheeosinophilic

countsofeachpatientwasusedto compare the two groups, thehyperinfected patientshad asignificantlylowerresponse (1.3 ± 1.2)than thenonhyperinfectedgroup (15.0 ± 9.8) (P < 0.02).

Lymphoproliferative responses were evaluated in four

individuals,

and none of them exhibited a high degree of

responsiveness to S. stercoralis antigens.

Regression analysis indicated thepresence of a relation-ship between totalIgEandspecificIgEvalues(r -0.7264, P

< 0.05). There was also a trend among patients with high specific IgE to have higher peripheral eosinophilia (r =

0.6325, P = 0.06). No correlation was

discovered

between

totalorspecific IgEandspecific IgGorbetweenspecificIgG andeosinophilia.

DISCUSSION

Theimmunological data obtained from this study suggest

that

parasite-specific IgG

antibodies

produced

inresponseto

S. stercoralis infection are present and can be usefully exploited for diagnosticpurposes alsoin immunosuppressed patients. These antibodies do not appear to represent an

indicator of the severity of disease or to

play

a central protective role against parasite dissemination. This is in

agreement with our findings with animal models, in which

specific

IgG titers did not have

predictive

value for the outcomeofanexperimental

infection

(8,

11).

Our series consisted of a small number

of patients,

and therefore the results should be interpreted cautiously. It

appeared, however,thateosinophilicresponseandthetotal IgElevelswerewellcorrelated withthe presenceof dissem-ination and withapoor

prognosis.

Patients with

hyperinfec-tion had significantly lower

peripheral eosinophilia

than

those without

dissemination,

and the two

patients

who

eventually succumbedtotheinfectionhad verylowtotal and

specific

IgE levels. A correlation between low

peripheral

eosinophilia and a poor prognosis in

disseminated

strongyloidiasishasbeen noted in other series(20, 31).This may be

eosinopenic

effect of

corticosteroids,

orit may reflect anincreased

migration

of

eosinophils

into

thetissues ina finaleffortto

impede

the

invading

larvae.

Eosinophils have been shown to have the

ability

to kill

helminthiclarvaein vitro

by

an

antibody-dependent

process (3, 22) and to be associated with in vivo

protection

from rat

schistosomiasis

(4).

Itis

possible

that in

strongyloidiasis

an

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interaction between eosinophils and IgE antibodies is nec-essary to prevent a widerdissemination of larvaefrom their normal autoinfectiouscycle. The behavior of the infection in our animal models seems to support this concept. In both experimentally infected steroid-treated monkeys (8) and dogs (11), fatal hyperinfection occurred in the presence of high specific IgG levels and high specific lymphocyte

reac-tivity, but only when the initial peripheral eosinophilia had subsided. However, low peripheral eosinophilia in the

ab-sence ofimmunosuppression does not appear to represent a

risk factorfor dissemination. In many series, 10 to 20% of

patients with chronic uncomplicated strongyloidiasis have been reportedtohavenormal peripheral eosinophilic counts

('5%)

and noevidence of hyperinfection (10, 14, 26). In immunocompetent patients, total IgE levels do not appear tocorrelate with the severity of disease. ForBritish former prisoners ofwarwithchronic strongyloidiasisand an

unusually high prevalence of larva currens, total IgE levels were normal in over 90% of the subjects (14), and in other

series they varied between50 and 70% (1, 8). In the present group, the two patients who died of disseminated

stron-gyloidiasis were those who had low total IgE levels and

minimal specific IgE responses. While it is important to

consider the smallnumbers of patients

studied,

thepossible protective role ofreaginic antibodies in S. stercoralis

infec-tionmay deservefurther investigation.

The results ofthe in vitro lymphoproliferative studies are

difficulttointerpret because none of the four patients tested

in thepresent study exhibited asignificant lymphoprolifera-tiveresponse toSS antigensin vitro. However, all patients were on high doses ofsteroids at the time of testing, and the

significance of lowresponses is therefore unclear.

The high specific IgE values observed for some patients who also had elevated titers of specific IgG antibodies may

indicate that inourRAST forstrongyloidiasisIgGantibodies didnot compete with orinhibit IgE binding to SS antigens.

This is further supported by the absence of correlation betweenspecific IgE and IgGresponses revealed by

regres-sion analysis of these responses.

Irrespective ofthe immunosuppression, all patients had

demonstrablelevels ofparasite-specific IgG. Although more

information is needed before the functional relevance of

these antibodiescan beevaluated,theirpracticalimportance

as a diagnostic tool should be stressed. It has been noted

that, particularly in patients withsevere disseminated

infec-tion, the nonspecific indicators ofparasitic disease

(periph-eraleosinophilia andelevated IgE) may be absent (20, 31),

and the present series further supports this finding. More-over, it is not unusual to encounter larva-negative stools in

patients with disseminated infection (23). In immunosup-pressedmonkeysexperimentallyinfected with S. stercoralis

wefoundoccasional negative stool larval counts even when

the intestinal worm burden was over 300,000 adults (18). This may be due to the fact thatduring dissemination more or even all rhabditiform larvae mature to the

tissue-penetrating filariform stage and reenter the intestinal wall

beforebeing passed with the feces.

Previously unsuspected S. stercoralis infections have been detected in Europe and in North America in patients whounderwent renaltransplants andcancerchemotherapy

(2, 7, 19, 25, 35). Most of these patients were natives of

countries where strongyloidiasis is highly prevalent, and

others hadaremotehistoryoftravel to such areas. Both the

BritishandtheAmericanreports of formerprisonersof war

(13,29) emphasize the unreliability of stoolexamination as a

diagnostic test as well as the widespread unawareness

amongpracticingphysicians of theimportance of this poten-tially fatal parasite.

Because of the side effects of thiabendazole (16, 32),

prophylactic treatment ofall candidates for immunosuppres-sion with a suggestive geographic history is controversial

and has not received wide acceptance (7; A. Bush, R.

Gabriel, S. J. Gatus,and J. G. Thornton, Letters, Br. Med.

J. 286:52). Extensive parasitological investigation of these

individuals, who for the most part are

asymptomatic,

is

unpractical and not cost effective. We suggest that the

ELISA may represent an excellent screening test for this

high-risk groupof patients. Furthermore, asindicated by the

resultsof our study, thisimmunoserologic test may havean important place in the diagnosis of disseminated infectionin

patients who are already immunocompromised.

The technology for the performance of the ELISA is

inexpensive and available in most clinicallaboratories. The

major limitingfactors are the difficulty of acquiringstandard positive sera and the lack of a ready source ofantigens. As described in this paper, ourELISAdepends on asingle high

positive standard serum to which test sera are compared. This is clearly not anoptimal method; it was used becauseof

constraints in obtaininglarge volumes ofserafrompositive

patients with S. stercoralis infection only. As our supply of

these sera hasincreased, standardpoolsof knownreactivity

are being prepared and will be made available to other investigators upon request.

As to the source of the antigens, S. stercoralis can be cultured from infected patients and maintained in

steroid-treated dogs for the production of filariform larval antigen (11). Several investigators have shown that ELISA (5, 28), indirect immunofluorescence tests (17), and the histamine

release assay (10) with Strongyloides ratti larval antigens are essentially as sensitive as those using S. stercoralis for the

detection of parasite-specificIgGand IgE antibodies. S. ratti is readily maintained in laboratory rats (27), and it may represent the most practical source of antigens for most

clinical applications ofthese serologic tests. ACKNOWLEDGMENTS

We areindebted to R. Quijano and D. H. Havlichek forreferring patients A and D. The invaluable technicalhelp of Theresa Huitger and JonnaMcRury is greatly appreciated. TheDocument Processing Area of the Department of Pathology and Laboratory Medicine providedcompetentsecretarial assistance.

This study was supported by the Medical Research Serviceof the Veterans Administration. P.D.W. is the recipient of a Clinical Investigator Award from the Veterans Administration.

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