CASE REPORT

Acute generalized exanthematous pustulosis in cysticechinococcosis: immunological characterization

C . C A N N I S T R A C I , I . L . L A P A R O L A , R . R I G A N O , * F . B A S S E T T I , E . O R T O N A , *

B . S A N T U C C I , M . P I C A R D O A N D S . S I R A C U S A N O *

S Gallicano Dermatological Institute, Via Chianesi, 53 00144 Rome, Italy

*Istituto Superiore di Sanita, Department of Immunology, Rome, Italy

Accepted for publication 7 November 2002

Summary We report the first case, to the best of our knowledge, of a woman suffering from cystic echino-

coccosis of the liver, who consequently developed urticaria and acute generalized exanthematous

pustolosis (AGEP). Serum immunoglobulin (Ig)E and IgG4 specific to Echinococcus granulosus

antigens were detected by immunoblotting. Furthermore, the intracellular cytokine analysis

revealed a prevalent T-helper 2 polarization. It can be reasoned that, while the presence of IgE

specific to various E. granulosus allergens may be responsible for the chronic urticarial manifesta-

tions, the detection of IgG4 specific for E. granulosus antigens, forming immunocomplexes, may be

related to the development of the AGEP.

Key words: acute generalized exanthematous pustolosis, echinococcosis, immunoblotting, intra-

cellular cytokine detection

Acute generalized exanthematous pustolosis (AGEP) is

a rare disease characterized by an acute extensive

eruption of numerous, small, nonfollicular sterile

pustules occurring atop widespread erythema, fever

above 38 �C and neutrophilic leukocytosis.

The mean duration of the pustules is 10 days and

most resolve within 15 days. The eruption is usually

followed within a few days by desquamation. The

histological findings are subcorneal pustules, papillary

oedema, subepidermal lymphohistiocytic perivascular

infiltrate with some polymorphonuclear neutrophils

and eosinophils, and focal keratinocyte necrosis.

AGEP is mostly caused by drugs, and less frequently

by viral infections, and exposure to mercury.1–6

The pathogenesis may depend upon an immunolo-

gical mechanism with the release of several cytokines

by T-helper (Th)-2 cells and the deposition of immu-

nocomplexes.

To our knowledge this is the first reported case of

AGEP occurring in a patient with cystic echinococcosis

of the liver. Cystic echinococcosis, caused by Echino-

coccus granulosus, like other helminthiases, manifests

hypersensitivity responses such as elevated serum

immunoglobulin (Ig)E and ⁄ or IgG4, eosinophilia and

mastocytosis.7–9

To investigate whether the cutaneous manifestations

observed in this patient were associated with parasit-

ism, we evaluated by immunoblotting the presence in

the patient’s serum of IgE and in particular IgG4

specific to E. granulosus antigens, a marker of growth

and disease progression. We also investigated the

intracellular cytokine expression in the patient’s T

lymphocytes to verify the polarization of T-cell

response.

Case report

A 70-year-old female, with a past history of intrahepatic

cysts of Echinococcus surgically removed in 1968, 1981

and 1986, was admitted to our S.Gallicano Dermato-

logical Institute for an episode of diffuse urticaria

2 years ago. A total body computed tomography (CT)

scan showed two cysts of the right lobe of the liver,Correspondence: Mauro Picardo.

E-mail: picardo@ifo.it

British Journal of Dermatology 2003; 148: 1245–1249.

� 2003 British Association of Dermatologists 1245

one a calcific cyst 3 cm in diameter, the other a liquid

cyst of 1 cm. Laboratory investigations, in accordance

with the standard protocol of urticaria, revealed an

IgE serum level 229 IU, erythrocyte sedimentation

rate 40, protein C reactive 1Æ95, a-acid glycoprotein

112, a high echinococcosis titre (1 : 1280). By

immunoblotting, IgE responses and Echinococcus anti-

gens were positive for hydatid fluid (antigen 5, antigen

B), protoscoleces proteins, while the IgG4 was negative.

Serological tests for infectious diseases and allergic

tests for inhalants, foods, drugs and chemicals all gave

negative results.

The patient did not take any drugs and there was no

personal or family history of atopy. She was treated for

2 months with oral antihistamines (cetirizine

10 mg day)1) and the cutaneous manifestations dis-

appeared.

In May 2000 the patient was again admitted for a

new episode of urticaria; she had been taking

antihistamines for 3 weeks (cetirizine 10 mg). Ultraso-

nography and CT scan showed two intrahepatic cysts;

the calcific cyst had not modified, while the liquid

cyst had increased in diameter (2 cm). Laboratory

investigations revealed echinococcosis at a titre of

1 : 2560.

Two days after admission, the patient presented a

generalized painful follicular pustular eruption with

diffuse erythema and a temperature of 39 �C (Figs 1

and 2). Microbiological examination of the pustules

presented no evidence of bacterial or fungal organ-

isms.

Therapy with albendazole, 400 mg twice daily and

oral prednisone 20 mg once a day for 5 days, was

administered and the skin eruption improved within a

few days. Albendazole therapy was continued for

6 weeks and three cycles were repeated without any

relapses of either urticaria or AGEP manifestations.

Materials and methods

Histopathology and direct immunofluorescence

Biopsy specimens were fixed in 10% buffered formalde-

hyde solution and embedded in paraffin. Paraffin

sections were prepared and stained with haematoxylin

and eosin, and cryostatic sections of the same cryopre-

served specimen used for the histological examination

were employed to detect immunoreactants in the lesion.

A fluorescein–isothiocyanate-labelled monoclonal

antibody directed against IgG4 (Sigma, St Louis, MO,

U.S.A.) was used in a direct immunofluorescence assay.

The antiechinococcus serum reactivity was carried out

by passive haemoagglutination (Alfa Biotech, Pomezia,

Italy).

Antigens

Sheep hydatid fluid (SHF) was collected from fertile

cysts for subsequent use as specific parasite antigen.

SHF was centrifuged at 10 000 g at 4 �C for 60 min

and protoscoleces were harvested and stored at )80 �C

until use. The supernatant was dialysed in phosphate

buffered saline pH 7Æ2, concentrated tenfold and

lyophilized. Recombinant antigens (antigen B, elonga-

tion factor 1 b ⁄ d, cyclophilin, heat shock protein 70)

were prepared as previously described.10

In brief, the fusion proteins were expressed in

Escherichia coli SG130009 cells, purified by affinity of

NI-NTA resin for the 6 · histidine tag and eluted with

urea under denaturing conditions according to the

supplier’s instructions (Qiagen, GmbH, Hilden,

Germany). Antigen B was obtained by polymerase

Figure 1. Clinical appearance. (A) A generalized follicular pustular

eruption with diffuse erythema. (B) An area of the skin eruption on

the back.

1 2 4 6 C . C A N N I S T R A C I et al.

� 2003 British Association of Dermatologists, British Journal of Dermatology, 148, 1245–1249

chain reaction (PCR) amplification of E. granulosus

genomic DNA using primers derived from the antigen B

sequence.11 After PCR, the fragments were run in 2%

agarose gel, purified with a Qiaex kit (Qiagen) as

instructed by the manufacturer, digested with restric-

tion enzymes and cloned in GST pGEx expression vector

(Amersham Pharmacia Biotech, Uppsala, Sweden). The

GST fusion protein was expressed and purified by

glutathione Sepharose 4B (Amersham Pharmacia Bio-

tech) using the batch method following the manufac-

turer’s instructions.

Immunoblotting

Immunoblotting, after 12% sodium dodecyl sulphate–

polyacrylamide gel electrophoresis in reducing condi-

tions, was performed as previously described.12 In brief,

the antigens were used at the concentrations of 3 lg

per lane and were revealed by human serum diluted at

1 : 50 for IgG4 detection and 1 : 10 for IgE detection.

Goat antihuman IgE peroxidase-labelled serum (Cappel,

Cochranville, PA, U.S.A.) and mouse monoclonal

antihuman-labelled serum (BioRad, Hercules, CA,

U.S.A.) were used as second antibodies. Goat antimouse

IgG peroxidase-labelled serum (BioRad) was used as the

third antibody.

Cellular immune response and intracellular cytokine

detection

Peripheral blood mononuclear cell (PBMC) proliferation

assay was done by the established procedure.13 Cyto-

kine expression was determined at intracellular level.

Whole blood samples were collected in sodium

heparin and activated with phorbol myristate acetate

(25 ng mL)1) plus ionomicin (1 lg mL)1) in the pres-

ence of Brefeldin-A (10 lg mL)1), which inhibits

intracellular transport of proteins following the proce-

dure of the current BDIS FASTIMMUNE Cytokine

System (Becton Dickinson, San Jose, CA, U.S.A.). After

an incubation of 4 h at 37 �C, 5% CO2, the samples

were lysed, permeabilized, stained with fluorescent-

conjugated anticytokine monoclonal antibodies (Bec-

ton Dickinson) and analysed on a FACScan flow

cytometer (Becton Dickinson) by a two-colour analysis

to determine the percentage of cells expressing intra-

cellular interferon (IFN)-c, interleukin (IL)-4 and IL-10.

In all experiments negative and positive samples for

each cytokine and samples from healthy nonatopic

subjects were included.

Results

A spongiotic pustule with neutrophils, a perivascular

polymorphous infiltrate and leukocytoclastic vasculitis

were found in the histological examination.

Direct immunofluorescence showed in situ deposits of

IgG4 confined to the vessel basal membrane. Qualitat-

ive analysis of IgE and IgG4 responses in the patient’s

serum determined by immunoblotting showed that IgE

recognized all hydatid antigens tested, whereas IgG4

was specific only for antigen B, SHF and protoscoleces

(Fig. 3A,B).

The patient’s PBMC showed a high specific prolifer-

ative response to all E. granulosus native and recom-

binant antigens used (Table 1).

Intracellular cytokine analysis of the patient’s T

lymphocytes showed that, in contrast to the results in

healthy nonatopic subjects, the proportion of IL-4 and

IL-10-producing cells (30% and 15%, respectively)

markedly exceeded the proportion of IFN-c-producing

cells (9%).

Figure 2. Histopathological findings. (A) Spongiotic pustule with

neutrophils, a perivascular polymorphous infiltrate and leukocyto-

clastic vasculitis. (B) Detail of leukocytoclastic vasculitis (haematoxylin

and eosin-stained sections; original magnification (A) · 40, (B) · 20.

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Discussion

Cystic echinococcosis is caused by the larval stage of

E. granulosus, which forms hydatid cysts containing

protoscoleces. The infection has a world-wide distribu-

tion, with higher prevalence in South America, Europe,

Northern Africa, Middle East, South-Central and East

Asia.14

Although all the organs may be affected, the liver is

the most common site of cyst development (50–75%)

with a less frequent pulmonary localization (30%).14

Specific cutaneous manifestations of hydatid disease,

characterized by fistulae running from hepatic cysts to

the skin and by metastatic cutaneous localization, are

rarely found. Nonspecific manifestations are pruritus

and urticaria.15–17

Immunological reactions to cystic echinococcosis

(sometimes related to cyst rupture) vary from hyper-

sensitive reactions, immunosuppressive effects and

complications associated with circulating immune

complexes.18

In our case two immune mechanisms were involved.

An IgE-mediated reaction occurred during the phase of

chronic urticarial manifestation followed by an IgG4-

mediated reaction, probably as a result of the prolonged

antigenic stimulation responsible for AGEP develop-

ment.

Several studies report that sera from patients with

cystic echinococcosis contain IgE specific to E. granulo-

sus hydatid fluid (antigen 5, antigen B) and to

constitutive protoscoleces proteins (elongation factor

1, cyclophilin, heat shock protein 70).19 The IgG4

response, the predominant histotype binding antigen

B,20 is associated with cystic development, growth and

disease progression and dominates the development

phase of the disease.21

In our patient we correlated the chronic urticarial

manifestations to the presence of IgE specific to various

E. granulosus allergens, while the clinical manifesta-

tions of the AGEP were correlated to immunocom-

plexes with IgG4 specific for antigen B, SHF and

protoscoleces.

The effect of parasite antigens on the immune

response of the patient is further supported by elevated

E. granulosus antigen-driven PBMC proliferation.

T cells control the synthesis of immunoglobulins.

Th1 and Th2 cell subsets interact through the secretion

of cytokines either by switching on (Th2, IL-4, IL-10)

or switching off (Th1, IFN-c) the synthesis of IgE and

IgG4 by B cells. T lymphocytes of the Th2 phenotype

are important in the pathophysiology of both allergic

disease and helminthic infections.22 In our patient,

the intracellular cytokine analysis demonstrated the

Figure 3. IgE (A) and IgG4 (B) reactivity in immunoblotting of the patient’s serum. Immunoblotting after 12% sodium dodecyl sulphate–

polyacrylamide gel electrophoresis of antigen B (lane 1), elongation factor 1 b ⁄ d (lane 2), heat shock protein 70 (lane 3), cyclophilin (lane 4),

sheep hydatid fluid (lane 5) and protoscoleces (lane 6).

Table 1. Proliferation of patient’s peripheral blood mononuclear cell

in response to Echinococcus granulosus antigens

Antigen Cpm (SD)

None 162 (30)

Sheep hydatid fluid 13000 (600)

Antigen B 1000 (150)

Elongation factor 1 b ⁄ d 1200 (200)

Heat shock protein 70 3300 (500)

Cyclophilin 2400 (300)

Cpm, Counts per minute (mean value of triplicate cultures); SD,

standard deviation.

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� 2003 British Association of Dermatologists, British Journal of Dermatology, 148, 1245–1249

presence of prevalent Th2 polarization, in accordance

with the profile of a humoral response.

In conclusion, we correlated AGEP to echinococco-

sis infection for three main reasons: (i) the patient had

not taken any drugs apart from cetirizine for 20 days,

and tests to verify food, drug and chemical allergies

were negative, as were the serological tests for

bacterial and viral diseases; (ii) the IgG4 positivity

for echinococcosis antigens observed in sera in skin in

association with the clinical manifestations of the

AGEP is probably correlated with the chronic anti-

genic stimulation; (iii) the good clinical response to

the therapy with albendazole improved the clinical

manifestations.

The allergic reactions reported during the course of

echinococcosis seem to be underestimated. However, it

should be stressed that the variability and severity of

the clinical manifestations in this parasitosis reflect

either the duration and intensity of the infection, or the

variety of human immune responses to parasite anti-

gens.

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