Antibody isotype responses to Schistosoma japonicum antigens in subjects from a schistosomiasis area with repeated praziquantel chemotherapy compared with a new endemic zone in Hunan Province, P.R. China

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(2002) 96,210-215

Antibody isotype responses to Schistosoma japonicum antigens in subjects from a schistosomiasis area with repeated praziquantel chemotherapy compared with a new endemic zone in Hunan Province, P.R. China ‘Hunan Institute and D. I’. McManu.G* of Parasitic Diseases, WHO Collaborating Centre for Research and Control of Schistosomiasis in Lake Regions, Huabanqiao Road, Yueyang, Hunan 414000, China; 2Molecular Parasitology Laborato y, Tropical Health Programme, Australian Centre for International and Tropical Health and Nutrition, The Queensland Institute of Medical Research and the University ‘Jiangsu Institute of Parasitic Diseases, of Queensland, 300 Herston Road, Herston, Brisbane, Qld 4006, Australia; Meiyuan, Wuxi, Jiangsu 214064, China

Y. Li’, D. B. Yu’, Y. S. Li’J, X. S. Luo’, Y. S. Liang3, P. B. BartleyZ

Abstract To demonstrate the dynamics of specific antibody isotypes against schistosome adult worm (AWA) and soluble egg (SEA) antigens, we evaluated (in 1999-2000) 112 subjects infected with Schistosoma japonicum from 2 regions of Hunan Province, China. Fifty-eight subjects were from Area A, a well-known endemic area with repeated chemotherapy. Area B (n = 54) is a new endemic focus in another part of the same province. Serum samples were collected prior to praziquantel (PZQ) chemotherapy, and at 2 and 12 months post-treatment. IgM, IgA, IgG, IgG,, IgG, and IgE antibodies to AWA and SEA were measured by quantitative enzyme-linked immunosorbent assay (ELISA). Pre-treatment antibody isotype levels from Area A, except IgA against AWA and SEA, were significantly higher than those from Area B. In response to chemotherapy, most antibody isotype levels fell or remained stable. However, in Area A there was a significant increase in the IgA, IgE and IgG, responses to AWA 2 months after PZQ-which fell to approach pre-treatment levels by 12 months. A similar response was seen in Area B with IgE and IgG, to AWA. Levels of all AWA-specific IgE and IgG, were significantly higher in subjects from Area A compared with Area B at all time-points. AWA-IgE levels demonstrated significant linear correlations with age and number of previous PZQ treatments in Area A only. All SEA-specific isotypes in both areas fell significantly in response to treatment-except IgE, which remained stable in both areas. All SEAspecific isotype levels (except IgA) were significantly higher from Area A at baseline. This significant difference was maintained through 12-months follow-up for IgE, IgGz and IgG, only. This study suggests that multiple episodes of schistosome infection may be required to generate antibody isotype levels that have been associated with resistance to re-infection in other studies. Further, a surrogate marker of successful chemotherapy (AWA-IgG4) performed less effectively in patients with previous treatment courses. Keywords: China

schistosomiasis, Schistosoma japonicum, chemotherapy, praziquantel, antibodies, isotype, IgE, IgG4,

Introduction The control of schistosomiasis japonica remains an imuortant uublic health nrioritv in China. The Yanatze Ri;er Valley and Dongting Lake regions of H&an Province are amongst the most severely affected areas (Yu et al., 1999; LI et al., 2000). Praziquantel chemotherapy of infected individuals and mass administration to the high-risk populations in endemic areas are critical components of all schistosomiasis control programmes worldwide (WHO, 1993). Previous serological studies of schistosomiasis patients (SHAKER et al., 1987; WILKINS et al., 1991; GRYSEELS et al., 1994; RAMIREz et al., 1996) have demonstrated that antibody isotype profiles are altered by praziquantel (PZQ) chemotherapv and that patient age mfluences this change (GRoGA&-~~ al., 1996; MuTAPI et al.. 1998). Re-infection. intensitv of infection (worm or egg burden), or duration of infection prior to therapy may influence antibody isotype profiles and therefore confound such studies in endemic areas. Here, we describe an evaluation of egg- and adult worm-specific antibody isotypes in schistosomiasis patients from 2 Chinese communities before and after PZQ therapy. One of these communities is a newly identified schistosomiasis-endemic area with little if any previous PZQ use. The other is a well-known endemic area with extensive previous PZQ exposure. Full details of the field survey of both areas, designed to compare the efficacy of PZQ against Schistosomajuponicum and to explore epidemiological factors that might affect the effectiveness of treatment, have been described (Yu et al., 2001).

*Author for correspondence; e-mail [email protected]

Materials

and Methods

Areas and population

Area A (Nanti and Chefan villages, Hanshou county) is in the western part of Dongting Lake in Hunan Province. It has a population of 2050 and has been a recognized schistosomiasis-endemic region for 50 years (Yu et al., 2001). The prevalence of S. japonicum infection in 1995 was about 20%. The area is adjacent to an extensive lake beach zone which provides an ideal habitat for Oncomelunia sp. snails. The climate is subtropical in nature with high rainfall and seasonal flooding. There are 2 distinct transmission seasons for cercarial shedding bv the snails: Mav-Iulv when the floods rise, and September-October -when the floods recede (LI et al., 2000). Most villagers are either tishermen or farmers (chiefly rice and cotton). There is virtually no reticulated (piped) water and open latrines are used by all. Annual PZQ chemotherapy to both humans and domestic animals commenced in 1987. Area B (Taipinqiao and Xiangtang villages, Hetang District, Zhuzhou City) is situated in the foothills of a mountainous region about 20 km from Zhuzhou City in Hunan Province. It has a population of 2180. Oncomelunia snails are distributed in the canals surrounding residential quarters and in ditches of paddy fields in the area. The majority of the residents are engaged in the production of rice and other economic crops. The climate and transmission seasons are similar to-those of Area A. S. iaponicum infection was first recorded in 1998 (XIAO”etaZ., 1998) and it is likely that S. juponicum was only recently introduced. A population survey with abdominal ultrasound demonstrated onlv rare instances of early-stage hepatic disease from chronic schistosomiasis KHEN et al.. 2000). Furthermore. the Oncomelania sp.‘snails in this region are distinct morphologically from those in well-defined long-term en-

ANTIBODY

ISOTYPES

AND

CHEMOTHERAPY

IN SCHISTOSOMIASIS

demic areas (XNO et al., 1998). PZQ chemotherapy was first used in this area in 1998 to treat stool-eggpositive cases. Subject participation

Informed consent was obtained from all participants following a detailed description of the purpose and potential benefits of the study. This study was approved by the Ethical Review Committee in the Hunan Institute of Parasitic Diseases. All subjects were interviewed to obtain demographic data, and information on occupation, educational status, previously diagnosed S. japonicum infection and previous PZQ treatments. Parasitological

examination

Baseline stool examinations commenced in November 1999, approximately 1 month after the end of the schistosomiasis transmission season. For stool examinations, 4 Kato-Katz slides were first prepared from a single stool sample with a total amount of 167 mg of et al., 1972). S. juponicum faeces per subject (KATZ eggs were counted 24 h after preparation by 2 experienced laboratory technicians from Hunan Institute of Parasitic Diseases: 10% of the slides were selected at random for quality assurance review. Eggs per gram faeces (epg) were calculated from the total number of eggs of the 4 slides multiplied by a factor of 6 (YU et al., 2001). The follow-up stool examinations were collected at 2 and 12 months after the baseline stool examination Praziquantel

treatment

After the initial stool examination, a single oral dose of 40 mg/kg PZQ (Shin Poong Pharm. Co. Ltd, Republic of Korea) was offered to the stool-egg-positive cases without charge. Those still stool-egg-positive at the end of this studv were offered PZQ re-treatment. The tablets were swallowed under medGa1 supervision and treated subjects were observed for 30 min to monitor side-effects (Yu et al., 2001). Serum collection and storage

A total of 112 infected individuals (58 from Area A and 54 from Area B) for this study were randomly selected from those (n = 236) stool-egg-positive at baseline (Yu et al., 2001) prior to chemotherapy. Blood was collected from the patients who became egg-negative-86 of the 112 initial subjects (Area A, 35; Area B, 51)-according to faeces sampling at the 2-months follow-up. A 12-month follow-up serum specimen was collected from 104 of the initial 112 subiects (54 from Area A and 50 from Area B) who still were egg-negative at this time. Additionally, 25 control samples were collected from healthy subjects from another region of Hunan Province where S. japonicum is not endemic. All sera were separated within 12 h of blood collection and stored at -70°C until analysed. Antigen preparation

Adult worm antigen (AWA) and soluble egg antigen (SEA) were prepared according to methods reported earlier ELI et al.. 1999a). In brief, adult S. iaponicum worms ‘(Chinese~Dongting strain) were collected from rabbits by perfusion of the hepatic portal system with a balanced salt solution, Eggs were recovered from rabbit livers 6 weeks after infection with 2000 cercariae. The washed worms and freeze-dried eggs were then homogenized separately in 0.3 mol pH 7.4 phosphate buffer in an ice-bath and ultracentrifuged at 100 000 g for 1 h at 4°C. The protein concentrations of the supernatant were determined by UV/VIS 751-G. The prepared AWA and SEA were stored at -20°C until use. Immunoassays Quantitative enzyme-linked immunosorbent assays (ELISAs) were performed using AWA and SEA as

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JAPONICA

antigen sources. The ELISA protocol followed procedures described previously (WEBSTER et al., 1997~; LI et al., 2001). Optimum incubation conditions and reagent dilutions were determined by chequerboard titration. Anti-human isotype antibodies conjugated with biotin were used to auantifv the serum AWA- and SEA-specific IgA, IgE, IgM, IgG, IgGz and IgG4 antibody concentrations. ELISA plates (MGOF-MBImmuno As>ay-Plate, Biomat Inc.) were coated with AWA or SEA (10 ug/mL), in 50 mM carbonate buffer (pH 9.6), and stored overnight at 4°C. Plates were washed 3 times with phosphate-buffered saline (pH 7.6) containing 0.05% Tween-20 (PBST), and blocked by 0.3% (w/v) bovine serum albumin (BSA) in PBS. The plates were washed 3 more times with PBST. The sera were diluted to 1:200 with 0.3% (w/v) BSA for the detection of IgG and IgM, to 1: 100 for IgG4 and IgA, and to 1: 10 for IgE and IgGz. After addition of diluted sera, plates were incubated at 37°C for 2 h. The plates were washed with PBST and the appropriate biotinconjugated anti-human globulin was added (mouse anti-human IgG,, IgG4 or goat anti-human IgA at 1:2000, goat anti-human IgE at 1: 1000, or IgG and IgM at 1: 10 000). After incubation for 1 h at 37°C the plates were washed in PEST and then incubated with Avidin-HRP (1: 1500, PharMingen Inc., USA) for 1 h at 37°C. The assays were developed with 3,3’,5,5’tetramethvlbenzidine (TMB peroxidase substrate, Sigma). Optical density (GD) values were read at 450 nm usinn a Bio-Rad EL-3 11 micro-mate reader. Internal standards were employed to m&imize plate-to-plate variation. analysis EpiInfo version 6.04b (Centers for Disease Control and Prevention, USA, and World Health Organization, Switzerland) was used for data entry. Statistical Package for Social Sciences version 10.0 (SPSS 10.0, SPSS Inc., 1999) was used to perform all descriptive and inferential statistical analyses. Levels of antibody isotypes in defined groups were expressed as arithmetic means of OD values. Student’s t test was utilized to test the differences between means. Pearson’s x2 was used to evaluate the statistical significance of differences in frequencies. The Pearson correlation coefficient was used to measure associations between variables. Statistical

Results Prevalence of infection Stool soecimens were submitted from 1129 residents from Area A and 1298 subjects from Area B. The prevalence of infection in Area B (8.17%, 106/1298) was significantly lower than Area A (10.98%, 124/ 1129; x’(1d.f.) = 5.58, P = 0.02). Demographic sub$cts

profile and history of chemotherapy

of infected

Demographic details of the 112 subjects available for follow-up are displayed in Table 1. Subjects were mostly male (79.3% for Area A and 53.7% for Area B) and predominantly rice farmers (74.1% for Area A and 63.0% for Area B). There were no differences in agegroup or infection intensity between the infected cases selected from the 2 areas. As expected, subjects from Area A had significantly greater PZQ exposure with an average of 3.58 treatments, compared with 0.17 times among individuals in Area B [t(llOd.f.) = 5.324, P < 0~0001]. Evolution

of antibody

isotypes

In general, the levels of antibodies against AWA and SEA 12 months post-treatment had decreased, and the patterns of declining levels of antibodies were similar in Area A and Area B (Figure). The levels of antibodies measured at 450 nm are reported in Table 2.

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Y. LI ETAL.

Table 1. Demographics and S. japonicum infection intensity subjects selected from Areas A and B of Hunan Province Area B (n = 54)

Area A (n = 58)

profiles

for the

Total (n = 112)

Variables

N

%

N

%

N

%

Gender Male Female

46 12

79.3 20.7

29 25

53.7 46.3

75 37

67.0 33.0

CO.001

18 40

31.0 69.0

24 30

44.4 55.6

42 70

37.5 62.5

>0.05

47 8 3

81.0 13.8 5.2

46 6 2

85.2 11.1 3.7

93 14 5

83.0 12.5 4.5

Age (years) <20 a20 Intensity

P value

(epg)

Cl00 101-400 2401

>0.05

fvalues were based on Pearson’s x2 test (Area A vs Area B). epg, Eggs per gram faeces.

Area A 2.4

2.4

AWA

T

2.2

l-z--i

2.0

2.0

1.8 1.6 g 1.4

1.8 1.6 1.4

g

1.2

1.2

;

1.0

1.0

0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

SEA

-

2.2

0.0

IgG

IgkI

IgA

IgE

IgG2

IgG4

W

IgM

W

W

IgG2

IgG4

Area B 2.4

2.4

AWA

2.2

2.2

2.0

4 g * 8

1.8 1.6 1.4 1.2 1.0 0.8

2.0

TI

IT

1.8

SEA

TT T II IT

1.6 1.4 1.2 1.0 0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

IgG

IgM

IgG2

Figure. Profiles of antibody levels (mean f SD) and post-treatment (Hunan Province). Area A miasis japonica. Area B is a new endemic focus 3, 12 months post-treatment with praziquantel;

IgG4

follow-up.

W

I@

Id

W

IgG2

IgG4

in the patients from Area A and Area B against S. juponicum AWA and SEA at preis a well-known endemic area with repeated previous chemotherapy for schistosoin another part of the same province. 1, Pre-treatment; 2, 2 months post-treatment; AWA, adult worm antigen; SEA, soluble egg antigen.

1. Pre-treatment baseline. Mean OD values from subhigher than values jects in Area A were significantly obtained from Area B for all antibody isotypes except anti-AWA IgA and anti-SEA IgA. In both areas, levels of all pre-treatment serum antibodies were significantly higher than those of control sera from the non-endemic area (P < 0.05). 2. Two-month

0.0

In Area B, anti-AWA

IgM

and IgA levels decreased significantly at 2 months posttreatment when compared with baseline values (P < 0.05). In both areas, the observed small changes in levels of anti-SEA IgG, IgM, IgA and IgGz and antiAWA IgG (and IgA in Area B) were not statistically significant. Relative to Area A, antibody isotype levels were significantly lower in subjects from Area B with the exception of anti-AWA IgGz and anti-SEA IgM,

IgA and IgE.

ANTIBODY ISOTYPES AND CHEMOTHERAPY

IN SCHISTOSOMIASIS JAPONICA

213

Table 2. The levels (ELISA, OD 450 nm) of antibody isotypes against S. juponicum and post-treatment in the individuals from Areas A and B of Hunan Province Pre-treatment Antibodies

2 months post-treatment

Controls (n=25)

Area A (n=%)

Area B (n=54)

P

Anti-SEA

IgM IgG

0.54 0.50 0.31 0.42

1.95 1.86 0.26 0.39

1.70 1.35 0.41 0.84

*,*

Anti-SEA

IgA IgE

0.330.16 0.08 0.06

0.33 1.220.30 0.23

0.21 1.15 0.26 0.38

;

Anti-SEA IgGz Anti-AWA Anti-SEA IgG4 IgG

0.06 0.08 0.20 0.18 0.18 0.12

1.53 0.63 0.84 1.59 051 0.38

0.77 0.71 0.51 1.20 0.59 0.44

** *,;

Anti-AWA Anti-AWA

IgM IgA IgE

0.48 0.32 0.30 0.04 0.08 0.20

1.97 0.32 0.35 0.89 0.45 0.28

1.49 0.59 0.96 0.18 0.41 0.27

** ;

Anti-AWA

IgG, IgG2

0.19 0.05 0.16 0.05

0.72 0.15 0.48 0.17

0.51 0.120.22 0.48

z

Area A (n= 35)

Area B (n=51)

1.74 151 1.17 0.18 1.01 0.96 1.63 157 1.06 0.70 0.24 0.89

1.32 1.57 1.14 0.18 0.58 0.66 1.24 0.80 1.26

0.39 0.35 0.36 0.22 0.57 0.50 0.33 0.39 0.35 0.55 0.17 0.52

AWA

and SEA pre-

12 months post-treatment P

0.76 0.42 0.45 0.32 0.58 0.47 0.63 0.39 0.51

** ** ;I

0.47 0.31 0.20 0.25 0.58 0.47

*

1%

*’

Area A (n= 54)

Area B (n=50)

P

0.82 0.17 0.63 0.13 0.77 0.30 0.30 0.18

0.74 0.30 055 0.24 0.72 0.17 0.18 0.25

;

0.230.29 0.42 0.44 0.86 0.22 0.76 0.94 0.20 0.23

0.11 0.24 0.90 0.86 0.92

0.16 0.36 0.34 0.41 0.19

* * ;

0.46 0.45 0.07 0.10 0.45 0.40

0.10 0.20 0.14 0.11 0.10 0.25

;; **

Values in each category are means followed by standard deviation. Area A is a well-known endemic area with reaeated orevious the motherapy for schistosomiasis japonica; Area B is a new endemic focus in another part of the same province. * ’ AWA, adult worm antigen; SEA, soluble egg antigen. **P < 0.01; *P < 0.05; -, P > 0.05.

Anti-AWA IgE levels in subjects from both areas increased significantly post-treatment (P < 0.05) particularly in Area A. There were also small but insignificant increases in anti-SEA IgG,, anti-AWA IgG, and IgG4 levels. All isotype levels remained significantly higher than those of control subjects from the nonendemic area (P < 0.05).

Discussion The identification of Area B as a recent endemic focus for schistosomiasis has facilitated further insights into the human humoral immune response against S. japonicum. Area B can be regarded as a true ‘new’ endemic area (as opposed to newly identified) because of the absence of any historical cases of disease that could reasonably be attributed to S. japonicum, and because all egg-positive subjects were free of clinical symptoms. The absence of ultrasound evidence of significant hepatic fibrosis in Area B supports this contention (CHEN et al., 2000). It is widely accepted that anti-AWA IgE may be a significant antibody response associated with protection against schistosome infection. Anti-AWA IgE levels correlate negatively with intensity of re-infection (DUNNE et al., 1992; DEMEURE et al., 1993; GROGAN et al., 1997). There are positive correlations between anti-AWA IgE (before and after PZQ therapy) and increasing age (DUNNE et al., 1992; GRYSEELS et al., 1994; GROGAN et al., 1996; WEBSTER et al., 1997b; ZHANG et al, 1997) with the implication being that IgE may play a role in the demonstrated age-dependent immunity to schistosomiasis. This study has demonstrated that PZQ chemotherapy of S. japona’cunz infection is associated with a corresponding rise in anti-AWA IgE antibodies in the

3. Twelve-month follow-up. All antibody isotype levels except anti-AWA IgA and anti-SEA IgE had declined significantly at the 12-month follow-up (P < 0.05) compared with the 2-month follow-up. Antibody isotype levels in this final sampling period were significantly below baseline levels (P < 0.05) in both areas except for anti-SEA IgE and anti-AWA IgA, IgE and IgGz. Levels of anti-SEA IgE, IgGz and IgG, and antiAWA IgE and IgG4 remained significantly higher in Area A (Table 2). In Area A, the levels of anti-AWA IgE at baseline and at both follow-up times correlated positively and significantly with age, number of previous PZQ treatments and levels of anti-AWA IgG4 (Table 3). In contrast, the only significant correlation with antiAWA IgE in Area B was a positive correlation with anti-AWA IgG, .

Table 3. Correlation coefficients (r) among the levels of anti-S. joponicum AWA IgE with age, previous treatments and the levels of IgG, in the patients from Areas A and B of Hunan Province Levels of anti-AWA (OD 450 nm)

I@,

Age

IgE

Previous treatments

r

P

r

P

r

P

Area A Pre-therapy 2 months post-therapy 12 months post-therapy

0.423 0.373 0.357

** * **

0.470 0.387 0.602

** * **

0.469 0.326 0.508

** * **

Area B Pre-therapy 2 months post-therapy 12 months post-therapy

-0.096 -0.237 0.101

-

0.516 0.613 0.505

** ** **

0.242 0.269 -0.098

-

AWA, adult worm antigen. **P < 0.01; *P < 0.05; -, P > 0.05.

214

short term in both Areas A and B. This rise might be due to the booster effects caused by released worm antigen after therapy. This view is consistent with some previous studies (EVENGARD et al., 1988; GROGAN et al., 1996; RAMIREZ et al., 1996; WEBSTER et al., 1997a; MUTAPI et al., 1998; NAUS et al., 1998) but not others (SHAKER et al., 1987; BOCTOR & PETER, 1990).

However, this response dropped below baseline in the newly identified endemic area but remained significantly elevated in the established endemic area after 12 months. There was significant positive correlation between age and levels of anti-AWA IgE in sera from subjects of Area A but not Area B. In thelong-established endemic focus. anti-AWA IaE levels also correlated nositivelv with me number of-previous PZQ treatments.-We were unable to demonstrate this correlation in another endemic region of Hunan Province (LI et al., 1999a) althouah we showed that anti-AWA IaE levels did correlate with resistance to re-infection by S. japonicum (LI et al., 1999b). The significant positive correlationin the long-established endemic- area only-between nrevious PZO treatments and anti-AWA IaE suaaests ‘that multiple-infections are required to generate-&tiAWA IgE levels that are potentially protective. These increases might also be the result of booster effects caused by released worm antigens. Because of its rapid disappearance following successful chemotherapy, some authors have viewed anti-AWA IgG4 as having the potential to be a surrogate marker for the efficacy of chemotherapy (BOCTOR & PETER, 1990; ROSS et al., 2001). In this study, the anti-AWA IgG, response to chemotherapy in the newly endemic area was consistent with this hypothesis. However, levels of anti-AWA IgG, 12 months post-therapy in the subjects from Area A (long-established focus and multiple treatments) remained significantly higher than in uninfected control individuals. This finding implies that the utility of antiAWA IgG, as a marker for successful therapy in an endemic area may be restricted. All SEA-svecific isotvnes in both areas fell sienificantly in response to treatment-except IgE, which remained stable in both areas. All SEA-specific isotype levels (except IgM) were significantly higher from Area A at baseline. The anti-SEA IgE levels did not reflect the pattern seen for AWA. The Production of IaE is stimulated bv interleukin13 (ILr13) and IL-4,“and modulated by IL-12 and interferon-gamma (KING et al., 1993; MEDHAT et al., 1998). The production of IgG4, is also stimulated by IL-4. IgG4 may specifically inhibit the action of IgE. In this study the positive correlation between anti-AWA IgE and IgG, most likely reflects this well-documented interaction, and is consistent with other studies of schistosomiasis (ISKANDER et al., 1981; DEMETJRE et al., 1993; LI et al., 1999a). In summary, this study suggests that multiple episodes of schistosome infection may be required to generate antibody isotype levels associated with resistance to re-infection. Further, a surrogate marker (anti-AWA IgG,) of successful chemotherapy performed less effectively in patients with previous treatment courses. Acknowledgements This study was supported financially by the Third World Academy of Science (97-3 11 RGiBIOiAS and WHO Regional Office of Western Pacific. References Boctor, F. N. & Peter, J. B. (1990). IgG subclasses in human chronic schistosomiasis: over-production of schistosomespecific and non-specific IgG,. Clinical and Experimental Immunology, 82,574-578. Chen, M. S., Chen, J. L., Li, L., Shou, Z. H. & Zhang, W. L. (2000). Epidemiological investigation in a new endemic area of schistosomiasis japonica in the suburb of Zhuzhou City

Y. LI ETAL.

[in Chinese]. Chinese Journal of Schistosomiasis Control, 12, 238-239. -ti Demeure, C. E., Rihet, I’., Abel, L., Ouattara, M., Bourgois, A. & Dessein, A. J. (1993). Resistance to Schistosoma mansoni in humans: influence of the IgEIIgG, balance and IgG2 in immunity to reinfection after chemotherapy. Journal of Infectious Diseases, 168, 1000-1008. Dunne, D. W., Butterworth, A. E., Fulford, A. J., Kariuki, H. C., Langley, J. G., Ouma, J. H., Capron, A., Pierce, R. J. & Sturrock, R. F. (1992). Immunity after treatment of human schistosomiasis: association between IgE antibodies to adult worm antigens and resistance to reinfection. European Journal ofImmunology, 22,1483-1494. Evengard, B., Hammarstrom, L., Smith, C. I. E., Johansson, S. G. 0. & Linder, E. (1988). Subclass distribution and IgE responses after treatment in human schistosomiasis. Clinical and Extwimental ImmunoloPv. 73. 383-387. Grogan, J. L., Kremsner, P.c; van Dam, G. J., Metzger, W., Mordmuller, B., Deelder, A. M. & Yazdanbakhsh, M. (1996). Antischistosome IgG, and IgE responses are affected differentially by chemotherapy in children versus adults. Jounzal of Infectious Diseases, 173, 1242- 1247. Grogan, J. L., Kremsner, I’. G., van Dam, G. J., Deelder, A. M. & Yazdanbakhsh, M. (1997). Anti-schistosome IgG4 and IgE at 2 years after chemotherapy: infected versus uninfected individuals. 3oumal of Infectious Diseases, 176, 1344-1350. Gryseels, B., Stelma, F. F., Talla, I., van Dam, G. J., Polman, K., Sow, S., Diaw, M., Sturrock, R. F., DoehringSchwerdtfeger, E., Kardorff, R., Decam, C., Niang, A. M. & Deelder, A. M. (1994). Epidemiology, immunology and chemotheraov of Schistosoma mansoni infection in a recentlv exposed community in Senegal. Tropical and Geographic;1 Medicine, 46, 209-219. Iskander, R., Das, P. K. & Aalberse, R. C. (1981). IaG4 antibodies-in Egyptian patients withschistosomiasis. I&& national Archives ofAllergy and Immunology, 66, 200-207. Katz, N., Caves, A. & Pelegrina, J. (1972). A simple device for quantitative stool thick-smear technique in schistosomiasis mansoni. Revista do Instituto de Medicina Tropical de SZo Puulo, 14,397-400. King, C. L., Low, C. C. & Nutman, T. B. (1993). IgE production in human helminth infection. Reciprocal interrelationship between IL-4 and IFN-y. 3ournaZ of Immunology, 150,1873-1880. Li, Y. S., Ross, A. G. I’., Sleigh, A. C., Li, Y., Waine, G. J., Williams. G. 1.. Tanner. M. & McManus. D. I’. f1999a). Antibody iso&e responses, infection and re-infection for Schistosoma japonicum in a marshland area of China. Acta Tropica, 73, 79-92. Li, Y. S., Sleigh, A. C., Ross, A. G. I’., Li, Y., Williams, G. M.. Forsvth. S. 1.. Tanner. M. & McManus. D. I’. (1999b‘i. A i-year prospective study in China provides epidemiological evidence for resistance in human to re-infection with Schistosoma iaponicum. Annals of” Tropical Medicine and Parasitology, 93,629-642. Li, Y. S., Sleigh, A. C., Ross, A. G. P., Williams, G. M., Tanner, M. & McManus, D. I’. (2000). Epidemiology of Schistosoma japonicum in China: morbidity and strategies for control in the Dongting Lake region. InternationalJournalfor Parasitolopv. 30. 273-28 1. Li, Y. S., Sgigh,A. C., Ross, A. G. P., Li, Y., Zhang, X. Y., Williams, G. M., Yu, X. L., Tanner, M. & McManus, D. P. (2001). Human susceptibility to Schistosoma japonicum in China correlates with antibody isotypes to native antigens. Transactions of the Royal Society of Tropical Medicine and Hygiene, 95,441-448.- Medhat. A.. Shehata. M.. Bucci. K.. Mohamed. S.. Dief. A. D.* E.,‘Badary, S., Galal, H.; Nafeh, M. & King; C. L: (1998). Increased interleukin-4 and interleukin-5 production in response to Schistosoma haematobium adult worm antigens correlates with lack of reinfection after treatment. Journal of Infectious Diseases, 178, 5 12-5 19. Mutapi, F., Ndhlovu, P. D., Hagan, P. & Woolhouse, M. E. (1998). Changes in specific anti-egg antibody levels following treatment with praziquantel for Schistosoma haematobium infection in children. Parasite Immunolopv, 20, 595-600. Naus, C. W., van Dam, G. J., Kremsner,?.- G.; Krijger, F. W. & Deelder, A. M. (1998). Human IgE, IgG subclass, and IgM responses to worm and egg antigens in schistosomiasis haematobium: a 12 month study of reinfection in Cameroonian children. Clinical Infectious Diseases, 26, 1142- 1147. Ramirez, R. M., Ceballos, E., Alarcon de Noya, B., Noya, 0. & Bianco, N. (1996). The immunopathology of human schistosomiasis-III. Immunoglobulin isotype profiles and

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Ross, A. G. I’., Sleigh, A. C., Li, Y., Davis, G. M., Williams, G. M., Jiang, Z., Feng, Z. & McManus, D. I’. (2001). Schistosomiasis in the People’s Republic of China: prospects and challenges for the 2 1st century. Clinical Microbiology Reviews, 14, 270-295. Shaker, Z. A., Hassanein, H. I., Kamel, M. M., El-Bahairy, N. M., El-Kalouby, A. H. & El-Raziky, E. H. (1987). Effect of praziquantel on certain immune responses of schistosomal Egyptian patients. I. Changes of specific immunoglobulins. Parasitoloev Reviews. 73. 328-333. Webster, M., Fallog, I’. G., Gulf&d, A. J., Butterworth, A. E., Ouma, J. H., Kimani, G. & Dunne, D. W. (1997a). Effect of praziquantel and oxamniquine treatment on human isotype responses to Schistosonza mansoni: elevated IgE to adult worm. Parasite Immunology, 19, 333-335. Webster, M., Libranda-Ramirez, B. D., Aligui, G. D., Olveda, R. M., Ouma, J. H., Kariuki, H. C., Kimani, G., Olds, G. R., Fulford, A. J., Butterworth, A. E. & Dunne, D. W. (1997b). The influence of sex and age on antibody isotype responses to Schistosoma mansoni and Schistosoma japonicum in human populations in Kenya and the Philippines. Parasitology, 114, 383-393. Webster, M., Correa-Oliveira, R., Gazzinelli, G., Viana, I. C., Fraga, L. A. D., Silveira, A. M. S. & Dunne, D. W. (1997c). Factors affecting high and low human IgE responses to schistosome worm antigens in an area of Brazil endemic for Schistosoma mansoni and hookworm. American Journal of Tropical Medicine and Hygiene, S4,487-494. WHO (1993). The Control of Schistosomiasis: Second Repon of WHO Expen Committee. Geneva: World Health Organization, Technical Report Series, no. 830.

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Prevalence of hepatitis E virus antibodies among different groups the Amazonian basin D. Kiesslich, J. E. Rocha Jr and M. A. Crispim Department of Research, Blood Centre, Manaus, Amazonas, Brazil

in

Amazonas

Keywords: hepatitis E virus, antibodies, seroprevalence, blood donors, haemodialysis patients, Amazon Basin, Brazil

Sanitary conditions in the Amazonian basin are favourable for enterically transmitted diseases. However, no outbreak of hepatitis E virus (HEV) has ever been described in this region, only sporadic cases of antibody detection in symptomatic or asymptomatic people, in small-sample studies, as reported by FOCACCIA et al. (1995), PANG et al. (1995) and SOUTO et al. (1997). We studied the prevalence of HEV antibodies in randomly selected groups of 227 voluntary blood donors (184 males, 43 females), 192 haemodialysis patients (116 males, 76 females) and 100 pregnant women, aged 14-87 years, using an enzyme immunoassay test (Abbott, Germany) with recombinant antigens SG-3 and 8-5 derived from ORF2 and ORF3, respectively, of the Burmese HEV strain. HEV antibodies were found only in 1 male blood donor (0.44%; 95% confidence interval [95% CI] Address for correspondence: Dr D. Kiesslich, Department of Research, Amazonas Blood Centre, Av. Constantino Nery, n 4397 - Chapada, CEP 69050-002 Manaus, Amazonas, Brazil; phone +55 92 6564020, fax +55 92 238 6798, e-mail [email protected]

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Wilkins, P. I’., Maddison, S. E., Slemenda, S. B. & Tsang, V. C. (1991). Isotypic analysis of humoral immune responses in rhesus monkeys to an adult microsomal antigen of Schistosoma mansoni: an indicator of successful treatment. AmericanJourna1 of Tropical Medicine and Hygiene, 45, 629-635. Xiao, M. Q., Feng, X. C., Chang, W. Q., Song, G. L., Zhou, F. S. & Xie, W. (1998). Analysis of results of parasitological examination of 846 residents in suburb villages of Zhuzhou City [in Chinese]. Chinese Journal of Epidemiology, 19, 218-219. Yu, D. B., Ross, A. G. I’., Xie, M. S., Li, Y. S. & Chen, Y. (1999). Highlights of the World Bank loan schistosomiasis control program in China (1991-1998): a special focus on Hunan province. Southeast Asian Journal of Tropical Medicine and Public Health, 30, 657-663. Yu, D. B., Li, Y., Sleigh, A. C., Yu, X. L., Li, Y. S., Wei, W. Y., Liang, Y. S. & McManus, D. I’. (2001). Efficacy of praziquantel against Schistosoma japonicum: field evaluation in an area with repeated chemotherapy compared with a newly identified endemic focus in Hunan, China. Transactions of the Royal Society of Tropical Medicine and Hygiene, 95,537-541. Zhang, Z., Wu, H., Chen, S., Hu, L., Xie, Z., Qiu, Y., Su, C,., Cao, J., Wu, Y., Zhang, S. & Wu, L. (1997). Isotypic antibody responses of a population in an endemic area of schistosomiasis japonica and their epidemiologic significance. Southeast AsianJournal of Tropical Medicine and Public Health, 28, 268-273.

Received 9 May 2001; revised 30 July 2001; accepted for publication 2 August 2001

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(2002) 96,215

0.02-2.81) and 1 female haemodialysis patient (0.51%; 95% CI 0.03-3.31). Neither had a history of hepatitis or jaundice. The former was a first-time donor and had travelled to a rural area within the previous year. The later had started haemodialysis sessions 3 months previously. The reactive samples were then tested with inhibition assay with recombinant HEV antigens SG-3 and 8-5 (Abbott, Germany). No significant inhibition was observed with the SG-3 antigen, but the 8-5 antigen caused a significant inhibition of 82.9% and 73.3% in the samples, indicating specific antibodies. In spite of the favourable environmental conditions to spread HEV, we found a low prevalence of antibodies in our study, but these results should be interpreted with caution. In Brazil, some studies have found that HEV might be circulating in other regions, inducing sporadic cases of symptomatic and asymptomatic infection. Anti-HEV tests used in seroprevalence studies in non-endemic areas must be confirmed with highly specific tests to avoid unreliable results. Acknowledgements The authors thank Thomas Schulte from Abbott Diagnostika-Germany for inhibition test results. References Focaccia, R., Sette, H. Jr & Conceicao, 0. J. (1995). Hepatitis E in Brazil. Lancet, 346, 1165. Pang, L., Alencar, F., Cerutti, C. Jr, Milhous, W. I<., Andrade, A. L., Oliveira, R., Kanessa-Thasan, N., Macarthy, I’. 0. & Hoke, C. H. Jr (1995). Short-report: hepatitis E infection in the Brazilian Amazon. American Journal of Tropical Medicine and Hygiene, 52, 347-348. Souto, F. J. D., Fontes, C. J. F., Parana, R. & Lyra, L. G. C. (1997). Short report: further evidence for hepatitis E in the Brazilian Amazon. American 3oumal of Tropical Medicine and Hygiene, 57, 149-150. Received 21 August September 2001

2001;

accepted for

publication

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