Inlernnlioml Journalfor Printed in Great Britain
Parasirology
Vol. 21, No. I,pp.
17-21, 1991 0
0020-7519/91 $3.00 + 0.00 Perglmlon Press plc 1991 Awmdian Sociefyfir Parosirology
DETECTION OF ANTIBODIES TO ANGIOSTRONGYLUS CANTONENSIS IN SERUM AND CEREBROSPINAL FLUID OF PATIENTS WITH EOSINOPHILIC MENINGITIS CHUAN-MIN Department
of Parasitology,
YEN*
and ENG-RIN
CHEN
Kaohsiung Medical College, No. 100, Shih-Chuan Taiwan, Republic of China
(Received 7 November
1st Rd, Kaohsiung,
1989; accepted 28 June 1990)
.&b&MC-YEN
C.-M. and CHEN E.-R. 1991. Detection of antibodies to Angiostrongylus cantonensisin serum and cerebrospinal fluid of patients with eosinophilic meningitis. International Journalfor Parasitology 21: 17-21. Adult and young adult antigens of Angiostrongylus cantonensis were purified by immuno-affinity chromatography and used to detect antibody in serum and cerebrospinal fluid (CSF), by enzyme-linked immunosorbent assay (ELISA), in cases of human eosinophilic meningitis or meningoencephalitis. The levels of IgG, IgA, IgM and IgE antibodies to A. cantonensis in these patients were higher than levels in control subjects. Antibodies in patients detected against adult and young adult worm antigens of A. cantonensis did not differ significantly. Significantly higher IgM and IgE antibody levels were observed in serum compared with CSF from infected patients (Student’s t-test. P < 0.05). Both adult and young adult A. cantonensis antigens proved to be highly sensitive in ELISA for serum antibodies; however, the sensitivity was significantly lower in tests on CSF. INDEX
KEY
WORDS:
Angiostrongylus
cantonensis:
INTRODUCTION
eosinophilic
meningitis;
antibodies;
cantonensis antigen has been measured by in vitro lymphocyte proliferative responsiveness (Yoshimura & Soulsby, 1976). A highly specific elevation of the lymphocyte proliferation response has also been observed in naturally infected rats (Welch & Dobson, 198 1). Various immunodiagnostic techniques utilizing either crude or partial purified adult worm antigen have also been used to examine humoral antibodies in the diagnosis of the infection (Welch, Dobson & Campbell, 1980; Chen, 1986; Cheng, Sato, Chen & Otsuru, 1989). Furthermore, the enzyme-linked immunosorbent assay (ELISA) utilizing A. cantonensis antigens prepared from young adult worms recovered from the rat brain 3 weeks after infection has shown promise for the sero-diagnosis of angiostrongyliasis (Tharavanij, 1979; Cross & Chi, 1982). Immunoglobulin levels and antibody titres in sera and CSF from patients with eosinophilic meningitis have been previously investigated (Tungkanak, Sirisinha & Punyagupta, 1972; Chen, 1975); however, there is no information on specific antibody levels identified by immunoglobulin classes. The present study was conducted to detect these antibodies in different classes of immunoglobulin by ELISA against adult and young adult worm antigens.
EOSINOPHILIC
meningitis or meningoencephalitis has been associated with infections of Angiostrongylus cantonensis and hundreds of cases have been diagnosed in southern Taiwan in past decades (Chen, 1979). People rarely harbour adult parasites although rats carry sexually mature worms in their pulmonary arteries and heart. Juvenile worms, however, have been found in the eyes, brain and spinal cord of infected people. Patients who suffer from eosinophilic meningitis or meningoencephalitis rarely have a parasitologically confirmed infection; juvenile worms are difficult to recover following a spinal tap (Cheng, Sato & Chen, 1984; Punyagupta, 1979). Clinical manifestations and laboratory findings are unreliable in the diagnosis of this parasitic disease because of confusion with other central nervous disorders. In spite of the inconvenience and the high risks involved in a spinal tap, the discovery of eosinophil leucocytes in cerebrospinal fluid (CSF), with a history of exposure, helps in the diagnosis of angiostrongyliasis. Many studies have been concerned with the detection of immune responses induced during the migration of larvae, and cell-mediated immune response in experimentally infected rats to A.
MATERIALS *To whom all correspondence
ELISA;
Subjects. Forty-eight or meningoencephalitis
should be addressed. 17
AND METHODS
patients with eosinophilic were tested for antibodies
meningitis specific to
C.-M. YEN and E.-R. CHEN
18
A. cantonensis. Both serum and CSF were obtained from 27 patients. Fifteen patients provided only serum specimens while six others provided only CSF specimens. Two millilitres of blood and/or CSF were collected and centrifuged at 4” C, the supematants were kept frozen until tested. Ten serum specimens from healthy volunteers and 10 CSF specimens from hospitalized patients without any evidence of neurological diseases were used as controls. Preparation of antigen. The third-stage larvae of A. cantonensis were collected by artificial digestion of Achatina fulica as described elsewhere (Yen, Chen, Hsieh & Dow, 1987). Mice infected for 3 weeks were killed and young adult worms removed from the brain. Adult worms were obtained from rats on the eighth week of infection by dissection of the lungs and heart. Both juvenile and adult worms were washed thoroughly with PBS, pH 7.2 and distilled water, then lyophilized and stored at -20’C. Adult and young adult worm antigens were prepared according to a previous report (Yen, Chen & Hsieh, 1984). Theextraction was carried out by grinding dried worms with triethanolamine buffered saline in a glass tissue grinder. Extracted solution was thoroughly mixed with ether and then centrifuged to remove the lipid layer. Supernatant was collected after high speed centrifugation. Purification of antigen was performed according to Welch Jr Dobson (1978). The first peak was pooled and concentrated. This fraction was then passed through a series of columns containing Sepharose 4B-cyanogen bromide, each specifically coupled with prepared rat immunoglobulin against Toxocara canis, Ascaris lumbricoides and Clonorchis sinensis. The glycine-HCl eluted fraction was dialyzed against PBS and protein concentration was adjusted to 10 pg ml-‘. Determination of antibody. Serum and CSF antibody levels in IgG, IgA, IgM and IgE were assayed by micro-ELISA (Yen et al., 1984). In brief, antigen was diluted to a concentration of 5 fig ml-.’ and dispensed into the wells of microtitre plates (Falcon 3912) and incubated at 37-C for 1 h. After blocking with 1.0% bovine serum albumin and washing with PBS containing 0.05% Tween-20, diluted serum or CSF (a dilution of 100 for IgG, IgA and IgM, and 10 for IgE) was added and incubated for 1 h. Unbound components of serum or CSF were washed off. Horseradish peroxidase conjugated with goat anti-human immunoglobulin (IgG, IgA, IgM or IgE) serum (Nordic, The Netherlands) were then reacted with the specific antibodies in serum or CSF by incubation. Excess conjugate was removed by washing four times. Microtiter plates were sealed with polyvinyl chloride resin membrane during each incubation. Freshly prepared substrate solution (12 mg O-phenylenediamine in 0.1 M-citric acid phosphate buffer solution, pH 5.0 containing 5 ~1 H,O,) was added into each well. Optical density (O.D.) was read at 490 nm using a Titertek Multiskan (Flow Co.) after 15 min incubation at room temperature. RESULTS Detection
of serum antibody Levels of specific IgG, IgA, IgM and IgE antibodies to A. cantonensis adult and young adult worm antigens
in patients and controls, detected by ELISA, are shown in Figs. 1 and 2. Generally, the distributions of ELISA values of IgG, IgA,IgM and IgE antibodies in patients were higher than those of uninfected persons, although there were a few overlapping values. The mean ELBA values from specific IgG, IgA, IgM and IgE antibodies in patients detected with A. cantonensis
.:.
I_ .:.. :ii : L .:.
2: ::.
.;.
‘:
:.
$1
;I
.::’
:::
‘2.’ 2 .:.
;E
‘::
OL
EM IG
EM HS IN
EM IN
FIG. 1. Distribution of ELISA values serum from eosinophilic meningitis or patients (EM) and healthy subjects A. cantonensis adult worms antigen. (I-1-l) mean f s
EM HS _ IgE
in the detection of meningoencephalitis (HS) with purified (-) ‘Cut off’ line, o.
0' -EM HS W
-EM HS @A
-EM HS IqM
-EM HS hE
FIG. 2. Distribution of ELISA values in the detection of serum from eosinophilic meningitis or meningoencephalitis patients (EM) and healthy subjects (HS) with purified A. cantonensis young adult worm antigen. (-) ‘Cut OK line, (I-1-l) mean f s. o.
adult worm antigen were in general similar to the results detected with young adult worm antigen. However, these mean ELBA value determinations in patients were significantly higher than those in
Antibodies in angiostrongyliasis uninfected persons (Student’s t-test, P < 0.05). IgG antibody demonstrated the highest and IgM antibody demonstrated the lowest increasing rates of adult worm or young adult worm antigen-specific antibodies in patients compared to uninfected persons, as shown in Table 1.
19
!4I-
Ii
IC,
TABLE I-THE INCREASINGRATES (%) OF A. cantonensis ANTIGEN-SPECIFIC ANTIBODIES IN PATIENTSCOMPARED WITHCONTROLS
Specimens
Antigens
IgG
IgA
IgM
IgE
Serum
Adult worm
118 127
79 76
56 58
86 109
96 120
65 65
68 63
88 100
0t 1-
il
OE
..J, .
2 9
Young adult worm CSF
Adult worm
Young adult worm
$
:.
s 04
:
.:.
Detection of CSF antibody Specific IgG, IgA, IgM and IgE antibodies in CSF to adult and young adult worm antigens of A. cantonensis were detected by ELISA and are shown in Figs. 3 and 4. The distributions of ELISA values of IgG, IgA, IgM and IgE antibodies in the patients group, shown as results in detection of serum antibodies, were generally higher than those of the control group and significant higher mean ELISA values (Student’s t-test, P< 0.05) were also observed in the patients group. The increasing rates of antigenspecific antibodies in patients compared to the control subjects, as shown in Table 1, were also generally similar to the rates in serum of patients, although there 14-
1.2 -
:;
‘:’
.:.
i .
.
C,-
EM---
EM
IgG
CS
IgA
.T.’
:
‘.‘.‘::
..
.
.i
::: ..1 ... .>
CS
EM
EM
. . ; ..
CS
IgE
I@'
FIG. 4. Distribution of ELISA values in the detection of CSF from eosinophilic meningitis or meningoencephalitis patients (EM) and control subjects (CS) with purified A. cantonensis young adult worm antigen. (-) ‘Cut off’ line, (1-1-l) mean f s.D.
was a slight decrease. When the mean ELISA values of IgG, IgA, IgM and IgE antibodies from serum of patients obtained by ELISA were compared with those from CSF of patients, significantly higher values were found in detection of serum (Student’s t-test, P < 0.05).
TABLE 2-THESENSITIV~TYAND SPECIFICITYINTHEDETECTIONOF ANTIBODIESBY IMMUNOGLOBULIN CLASSES IN SERUMAND CSF OF EOSINOPHlLlCMENINGlTlSOR MENINGOENCEPHALITIS PATIENTSBY ELISA US~NGPURIFIEDADULTAND YOUNG ADULT A. cantonensis WORMANTIGENS
0.8 a
:
:
.:’
.... : .
:: ..
::.
Sensitivity and speciJicity The ‘cut off’ point among ELISA values tested for antibodies by classes was set up individually as
1.0 -
:1
G
:
Oi
:. ::: -I-:. J-J 1
4.:
‘.. .:
0.6 -
..:.. i ::. .: .:. . 4
Igs
Sensitivity
Specificity
Adult
Young adult
Adult
Young adult
90.5* 81.0 83.3 83.3
97.6t 81.0 83.3 78.6
100.0 80.0 80.0 90.0
90.0 80.0 80.0 90.0
75.8* 75.8 75.8 69.7
75.8t 72.7 75.8 69.7
100.0 80.0 80.0 80.0
90.0 80.0 80.0 80.0
Serum
:;I
0’ EM cs ---IgG
EM I@
cs
EM IW
CS
EM
CS
IgE
FIG. 3. Distribution of ELISA values in the detection of CSF from eosinophilic meningitis or meningoencephalitis patients (EM) and control subjects (CS) with purified A. cantonensis adult worm antigen. (-) ‘Cut off’ line, (I-I-I) mean f S.D.
IeG
IA
IgM IgE
CSF IgG
IgA IgM IgE
* Chi-square test, x2 = 3.0, P < 0.05. t Chi-square test, x2 = 10.3, P < 0.01.
20
C.-M. YEN and E.-R. CHEN
indicated in Figs. 14 to achieve critical positive reactions. Sensitivity and specificity in detection of antibodies for angiostrongyliasis were evaluated as described by Yen et al. (1984) and the data are shown in Table 2. The patterns of specificity in detection of serum and CSF antibodies against adult and young adult A. cantonensis antigen by ELISA were similar. However, the test for IgG antibody in serum, for both adult and young adult worm antigens, was greater than those of tests for IgA, IgM and IgE antibodies. The sensitivity in detection of serum IgG antibody was also significantly higher that that for IgG antibody in CSF (Student’s r-test, P < 0.05). DISCUSSION
Antigens from several stages of A. cantonensis have been used in immunological tests to diagnose angiostrongyliasis (see Tharavanij, 1979); however, the best immunodiagnostic antigens have yet to be determined. A polypeptide with a specific molecular weight, prepared from the third-stage larvae, was found to stimulate a considerable amount of antibody (Dharmkrong-AT & Sirisinha, 1983). Thus antigens from larvae may be important in the immunodiagnosis of A. cantonensis, particularly in early infection. However, the incubation period-between infection and symptoms-of the disease in humans is more than 2 weeks (Hung & Chen, 1988). By this time, the larvae have developed to the juvenile stage in the CNS prior to the onset of illness. Some reports indicate that juvenile worm antigen gives better immunodiagnosis of angiostrongyliasis while other reports prefer adult worm antigens (Chen, 1986; Cross & Chi, 1982; Sato, Otsuru, Asato & Kinjo, 1977; Cross, 1978). Here the sensitivity and specificity in the detection of antibodies with adult worm antigen appeared very similar to those for young adult worm antigens. However, the sensitivity in the detection of IgG antibody in CSF specimens was significantly less than that in sera. Thus, adult and young adult worm antigens can be used in sero-diagnosis of A. cantonensis infection by ELISA; serum, rather than CSF, better illustrates the status of the infection in the patient. The immunoglobulins of the serum and the CSF of patients were compared qualitatively and quantitatively with those in fluids from the control subjects. Total IgG, IgA and IgM concentrations in the CSF of patients with eosinophilic meningitis were significantly greater than those in CSF from normal patients (Tungkanak et al., 1972; Chen, 1975). The total IgG concentration from infected patients was significantly higher than that in the normal controls only in one case (Tungkanak et al., 1972); however, significantly higher levels of IgE and IgM were observed in patients in another study (Chen, 1975). Perhaps the small sample size and the variety of patients were reasons for these divergent observations. The increase of any class of immunoglobulin in patients could not truly represent the total immune response which might include nonspecific components that would not react with worm
antigens. The serum IgG, IgA, IgM and IgE antibodies which act against both A. cantonensis adult and young adult worm antigens were elevated significantly in this present study. The increasing levels of the four classes of immunoglobulins in the CSF specific to worm antigens corresponded with changes in the immunoglobulins in the serum. The increase of specific immunoglobulins to A. cantonensis in the CSF may also come via the blood by changes in the permeability of the blood-brain barrier. However, some of the evidence indicates that local immunoglobulin synthesis is involved in the central nervous system in response to an infection with A. cantonensis (see Yoshimura & Soulsby, 1976). In the present study, the increasing rate of antigenspecific IgG antibody in patients was greater than that for IgM antibody seen 1 month after symptoms of A. cantonensis infection. This may be the reason why there was a significant rise in IgG antibody in our patients. Evidence from in vitro studies on IgG antibody suggested that antibody-dependent cellmediated cytotoxicity (ADCC) was involved (Yoshimura, Uchida, Sato & Oya, 1983; Yen, C. M., Unpublished PhD thesis, Kaohsiung Medical College, Taiwan, 1988) as the main protective mechanism against infections with A. cantonensis. Parasites or their eggs in the intestine of hosts can stimulate immune responses in the mucosa to produce IgA antibody which appears in the serum and as secretory IgA antibody in the intestinal lumen (David, 1982). Why IgA antibody levels were elevated significantly in the serum and CSF of patients with angiostrongyliasis remains obscure. It is well known that high serum levels of IgE have been observed in many helminth infections (Kojima, Yokogawa & Tada, 1972; Genta, Ottesen, Poindexter, Gam, Neva, Tanowitz & Wittner, 1983; Chen & Yen, 1984; Chen, 1975). IgE antibody can induce ADCC and damage schistosomula (Capron, Bazin, Joseph & Capron, 1981; Kojima, Niimura & Kanazawa, 1987). Significantly higher antigen-specific IgE antibody levels were observed here in patients with angiostrongyliasis. To demonstrate whether IgE antibody also has the ability of ADCC in infection with A. cantonensis requires a further study to be undertaken. Acknowledgemenrs-The authors wish to thank Kao-Pin Huang, Departments of Parasitology and Pediatrics, Kaohsiung Medical College, for assistance in collection of serum and CSF specimens. We thank Wun-Yuen Dow, Department of Parasitology, Kaohsiung Medical College, for carrying out the ELBA. REFERENCES CAPRON M.,
BAZIN H., JOSEPHM. & CAPRON A.
1981.
Evidence of IgE-dependent cytotoxicity by rat eosinophils. Journal of Immunology 126: 1764-l 769. CHEN E. R. 1979. Angiostrongyliasis and eosinophilic meningitis in Taiwan: a review. In: Studies on Angiostrongyliasis in Eastern Asia andAustralia (Edited by CROSS J. H.), 57-73. Special Publication of the U.S. Naval Medical Research Unit No. 2, Taipei, Taiwan.
Antibodies
in angiostrongyliasis
CHEN E. R. & YEN C. M. 1984. Study on the control of zoonotic clonorchiasis (III). Human survey, immunodiagnosis and treatment. National Science Council Monthly (R. 0. C.) 11: 1401-1408. CHEN S. N. 1975. Blood and cerebrospinal fluid findings in eosinophilic meningitis and antibody to Angiostrongylus cantonensis. Bulletin of Institute of Zoology, Academy Sinica 14: 109-l 13. CHEN S. N. 1986. Enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Angiostrongylus cantonensis. Transactions of the Royal Society of Tropical Medicine and Hygiene 80: 398-405. CHENGC. W., SATO Y. & CHEN E. R. 1984. Epidemiological and clinical observations on eosinophilic meningitis and meningoencephalitis of children caused by infection with A. cantonensis in southern Taiwan. Ryukyu Medical Journal 7: l-9. CHENG C. W., SATO Y., CHEN E. R. & OTSURU M. 1989. Application of enzyme-linked immunosorbent assay (ELISA) in serodiagnosis of angiostrongyliasis, with special reference to the reactivity of the extracts from adult and young adult worms. Chinese Journal of Parasitology 2: 3246. CROSS J. H. 1978. Clinical manifestation and laboratory diagnosis of eosinophilic meningitis syndrome associated with angiostrongyliasis. Southeast Asian Journal of Tropical Medicine and Public Health 9: 161-170. CROSS J. H. & CHI J. C. H. 1982. ELISA for the qletection of Angiostrongylus cantonensis antibodies in pstients with eosinophilic meningitis. Southeast Asian Journ@ of Tropical Medicine and Public Health 13: 73-76. DAVID J. R. 1982. Immune effector mechanisms against parasites. In: Immunology of Parasitic Infections (Edited by COHENS. &WARREN K. S.), pp. 74-98. Blackwell Scientific Publications, Oxford. DHARMKRONG-AT A. & SIR~SINHA S. 1983. Analysis of antigens from different developmental stages of Angiostrongylus cantonensis. Southeast Asian Journal of Tropical Medicine and Public Health 14: 154-l 62. GENTA R. M., O~ESEN E. A., POINDEXTERR., GAM A. A., NEVA F. A., TANOWITZH. B. & W~~TNERM. 1983. Specific allergic sensitization to Strongyloides antigens in human stro
Parasirology
Vol. 21, No. I,pp.
17-21, 1991 0
0020-7519/91 $3.00 + 0.00 Perglmlon Press plc 1991 Awmdian Sociefyfir Parosirology
DETECTION OF ANTIBODIES TO ANGIOSTRONGYLUS CANTONENSIS IN SERUM AND CEREBROSPINAL FLUID OF PATIENTS WITH EOSINOPHILIC MENINGITIS CHUAN-MIN Department
of Parasitology,
YEN*
and ENG-RIN
CHEN
Kaohsiung Medical College, No. 100, Shih-Chuan Taiwan, Republic of China
(Received 7 November
1st Rd, Kaohsiung,
1989; accepted 28 June 1990)
.&b&MC-YEN
C.-M. and CHEN E.-R. 1991. Detection of antibodies to Angiostrongylus cantonensisin serum and cerebrospinal fluid of patients with eosinophilic meningitis. International Journalfor Parasitology 21: 17-21. Adult and young adult antigens of Angiostrongylus cantonensis were purified by immuno-affinity chromatography and used to detect antibody in serum and cerebrospinal fluid (CSF), by enzyme-linked immunosorbent assay (ELISA), in cases of human eosinophilic meningitis or meningoencephalitis. The levels of IgG, IgA, IgM and IgE antibodies to A. cantonensis in these patients were higher than levels in control subjects. Antibodies in patients detected against adult and young adult worm antigens of A. cantonensis did not differ significantly. Significantly higher IgM and IgE antibody levels were observed in serum compared with CSF from infected patients (Student’s t-test. P < 0.05). Both adult and young adult A. cantonensis antigens proved to be highly sensitive in ELISA for serum antibodies; however, the sensitivity was significantly lower in tests on CSF. INDEX
KEY
WORDS:
Angiostrongylus
cantonensis:
INTRODUCTION
eosinophilic
meningitis;
antibodies;
cantonensis antigen has been measured by in vitro lymphocyte proliferative responsiveness (Yoshimura & Soulsby, 1976). A highly specific elevation of the lymphocyte proliferation response has also been observed in naturally infected rats (Welch & Dobson, 198 1). Various immunodiagnostic techniques utilizing either crude or partial purified adult worm antigen have also been used to examine humoral antibodies in the diagnosis of the infection (Welch, Dobson & Campbell, 1980; Chen, 1986; Cheng, Sato, Chen & Otsuru, 1989). Furthermore, the enzyme-linked immunosorbent assay (ELISA) utilizing A. cantonensis antigens prepared from young adult worms recovered from the rat brain 3 weeks after infection has shown promise for the sero-diagnosis of angiostrongyliasis (Tharavanij, 1979; Cross & Chi, 1982). Immunoglobulin levels and antibody titres in sera and CSF from patients with eosinophilic meningitis have been previously investigated (Tungkanak, Sirisinha & Punyagupta, 1972; Chen, 1975); however, there is no information on specific antibody levels identified by immunoglobulin classes. The present study was conducted to detect these antibodies in different classes of immunoglobulin by ELISA against adult and young adult worm antigens.
EOSINOPHILIC
meningitis or meningoencephalitis has been associated with infections of Angiostrongylus cantonensis and hundreds of cases have been diagnosed in southern Taiwan in past decades (Chen, 1979). People rarely harbour adult parasites although rats carry sexually mature worms in their pulmonary arteries and heart. Juvenile worms, however, have been found in the eyes, brain and spinal cord of infected people. Patients who suffer from eosinophilic meningitis or meningoencephalitis rarely have a parasitologically confirmed infection; juvenile worms are difficult to recover following a spinal tap (Cheng, Sato & Chen, 1984; Punyagupta, 1979). Clinical manifestations and laboratory findings are unreliable in the diagnosis of this parasitic disease because of confusion with other central nervous disorders. In spite of the inconvenience and the high risks involved in a spinal tap, the discovery of eosinophil leucocytes in cerebrospinal fluid (CSF), with a history of exposure, helps in the diagnosis of angiostrongyliasis. Many studies have been concerned with the detection of immune responses induced during the migration of larvae, and cell-mediated immune response in experimentally infected rats to A.
MATERIALS *To whom all correspondence
ELISA;
Subjects. Forty-eight or meningoencephalitis
should be addressed. 17
AND METHODS
patients with eosinophilic were tested for antibodies
meningitis specific to
C.-M. YEN and E.-R. CHEN
18
A. cantonensis. Both serum and CSF were obtained from 27 patients. Fifteen patients provided only serum specimens while six others provided only CSF specimens. Two millilitres of blood and/or CSF were collected and centrifuged at 4” C, the supematants were kept frozen until tested. Ten serum specimens from healthy volunteers and 10 CSF specimens from hospitalized patients without any evidence of neurological diseases were used as controls. Preparation of antigen. The third-stage larvae of A. cantonensis were collected by artificial digestion of Achatina fulica as described elsewhere (Yen, Chen, Hsieh & Dow, 1987). Mice infected for 3 weeks were killed and young adult worms removed from the brain. Adult worms were obtained from rats on the eighth week of infection by dissection of the lungs and heart. Both juvenile and adult worms were washed thoroughly with PBS, pH 7.2 and distilled water, then lyophilized and stored at -20’C. Adult and young adult worm antigens were prepared according to a previous report (Yen, Chen & Hsieh, 1984). Theextraction was carried out by grinding dried worms with triethanolamine buffered saline in a glass tissue grinder. Extracted solution was thoroughly mixed with ether and then centrifuged to remove the lipid layer. Supernatant was collected after high speed centrifugation. Purification of antigen was performed according to Welch Jr Dobson (1978). The first peak was pooled and concentrated. This fraction was then passed through a series of columns containing Sepharose 4B-cyanogen bromide, each specifically coupled with prepared rat immunoglobulin against Toxocara canis, Ascaris lumbricoides and Clonorchis sinensis. The glycine-HCl eluted fraction was dialyzed against PBS and protein concentration was adjusted to 10 pg ml-‘. Determination of antibody. Serum and CSF antibody levels in IgG, IgA, IgM and IgE were assayed by micro-ELISA (Yen et al., 1984). In brief, antigen was diluted to a concentration of 5 fig ml-.’ and dispensed into the wells of microtitre plates (Falcon 3912) and incubated at 37-C for 1 h. After blocking with 1.0% bovine serum albumin and washing with PBS containing 0.05% Tween-20, diluted serum or CSF (a dilution of 100 for IgG, IgA and IgM, and 10 for IgE) was added and incubated for 1 h. Unbound components of serum or CSF were washed off. Horseradish peroxidase conjugated with goat anti-human immunoglobulin (IgG, IgA, IgM or IgE) serum (Nordic, The Netherlands) were then reacted with the specific antibodies in serum or CSF by incubation. Excess conjugate was removed by washing four times. Microtiter plates were sealed with polyvinyl chloride resin membrane during each incubation. Freshly prepared substrate solution (12 mg O-phenylenediamine in 0.1 M-citric acid phosphate buffer solution, pH 5.0 containing 5 ~1 H,O,) was added into each well. Optical density (O.D.) was read at 490 nm using a Titertek Multiskan (Flow Co.) after 15 min incubation at room temperature. RESULTS Detection
of serum antibody Levels of specific IgG, IgA, IgM and IgE antibodies to A. cantonensis adult and young adult worm antigens
in patients and controls, detected by ELISA, are shown in Figs. 1 and 2. Generally, the distributions of ELISA values of IgG, IgA,IgM and IgE antibodies in patients were higher than those of uninfected persons, although there were a few overlapping values. The mean ELBA values from specific IgG, IgA, IgM and IgE antibodies in patients detected with A. cantonensis
.:.
I_ .:.. :ii : L .:.
2: ::.
.;.
‘:
:.
$1
;I
.::’
:::
‘2.’ 2 .:.
;E
‘::
OL
EM IG
EM HS IN
EM IN
FIG. 1. Distribution of ELISA values serum from eosinophilic meningitis or patients (EM) and healthy subjects A. cantonensis adult worms antigen. (I-1-l) mean f s
EM HS _ IgE
in the detection of meningoencephalitis (HS) with purified (-) ‘Cut off’ line, o.
0' -EM HS W
-EM HS @A
-EM HS IqM
-EM HS hE
FIG. 2. Distribution of ELISA values in the detection of serum from eosinophilic meningitis or meningoencephalitis patients (EM) and healthy subjects (HS) with purified A. cantonensis young adult worm antigen. (-) ‘Cut OK line, (I-1-l) mean f s. o.
adult worm antigen were in general similar to the results detected with young adult worm antigen. However, these mean ELBA value determinations in patients were significantly higher than those in
Antibodies in angiostrongyliasis uninfected persons (Student’s t-test, P < 0.05). IgG antibody demonstrated the highest and IgM antibody demonstrated the lowest increasing rates of adult worm or young adult worm antigen-specific antibodies in patients compared to uninfected persons, as shown in Table 1.
19
!4I-
Ii
IC,
TABLE I-THE INCREASINGRATES (%) OF A. cantonensis ANTIGEN-SPECIFIC ANTIBODIES IN PATIENTSCOMPARED WITHCONTROLS
Specimens
Antigens
IgG
IgA
IgM
IgE
Serum
Adult worm
118 127
79 76
56 58
86 109
96 120
65 65
68 63
88 100
0t 1-
il
OE
..J, .
2 9
Young adult worm CSF
Adult worm
Young adult worm
$
:.
s 04
:
.:.
Detection of CSF antibody Specific IgG, IgA, IgM and IgE antibodies in CSF to adult and young adult worm antigens of A. cantonensis were detected by ELISA and are shown in Figs. 3 and 4. The distributions of ELISA values of IgG, IgA, IgM and IgE antibodies in the patients group, shown as results in detection of serum antibodies, were generally higher than those of the control group and significant higher mean ELISA values (Student’s t-test, P< 0.05) were also observed in the patients group. The increasing rates of antigenspecific antibodies in patients compared to the control subjects, as shown in Table 1, were also generally similar to the rates in serum of patients, although there 14-
1.2 -
:;
‘:’
.:.
i .
.
C,-
EM---
EM
IgG
CS
IgA
.T.’
:
‘.‘.‘::
..
.
.i
::: ..1 ... .>
CS
EM
EM
. . ; ..
CS
IgE
I@'
FIG. 4. Distribution of ELISA values in the detection of CSF from eosinophilic meningitis or meningoencephalitis patients (EM) and control subjects (CS) with purified A. cantonensis young adult worm antigen. (-) ‘Cut off’ line, (1-1-l) mean f s.D.
was a slight decrease. When the mean ELISA values of IgG, IgA, IgM and IgE antibodies from serum of patients obtained by ELISA were compared with those from CSF of patients, significantly higher values were found in detection of serum (Student’s t-test, P < 0.05).
TABLE 2-THESENSITIV~TYAND SPECIFICITYINTHEDETECTIONOF ANTIBODIESBY IMMUNOGLOBULIN CLASSES IN SERUMAND CSF OF EOSINOPHlLlCMENINGlTlSOR MENINGOENCEPHALITIS PATIENTSBY ELISA US~NGPURIFIEDADULTAND YOUNG ADULT A. cantonensis WORMANTIGENS
0.8 a
:
:
.:’
.... : .
:: ..
::.
Sensitivity and speciJicity The ‘cut off’ point among ELISA values tested for antibodies by classes was set up individually as
1.0 -
:1
G
:
Oi
:. ::: -I-:. J-J 1
4.:
‘.. .:
0.6 -
..:.. i ::. .: .:. . 4
Igs
Sensitivity
Specificity
Adult
Young adult
Adult
Young adult
90.5* 81.0 83.3 83.3
97.6t 81.0 83.3 78.6
100.0 80.0 80.0 90.0
90.0 80.0 80.0 90.0
75.8* 75.8 75.8 69.7
75.8t 72.7 75.8 69.7
100.0 80.0 80.0 80.0
90.0 80.0 80.0 80.0
Serum
:;I
0’ EM cs ---IgG
EM I@
cs
EM IW
CS
EM
CS
IgE
FIG. 3. Distribution of ELISA values in the detection of CSF from eosinophilic meningitis or meningoencephalitis patients (EM) and control subjects (CS) with purified A. cantonensis adult worm antigen. (-) ‘Cut off’ line, (I-I-I) mean f S.D.
IeG
IA
IgM IgE
CSF IgG
IgA IgM IgE
* Chi-square test, x2 = 3.0, P < 0.05. t Chi-square test, x2 = 10.3, P < 0.01.
20
C.-M. YEN and E.-R. CHEN
indicated in Figs. 14 to achieve critical positive reactions. Sensitivity and specificity in detection of antibodies for angiostrongyliasis were evaluated as described by Yen et al. (1984) and the data are shown in Table 2. The patterns of specificity in detection of serum and CSF antibodies against adult and young adult A. cantonensis antigen by ELISA were similar. However, the test for IgG antibody in serum, for both adult and young adult worm antigens, was greater than those of tests for IgA, IgM and IgE antibodies. The sensitivity in detection of serum IgG antibody was also significantly higher that that for IgG antibody in CSF (Student’s r-test, P < 0.05). DISCUSSION
Antigens from several stages of A. cantonensis have been used in immunological tests to diagnose angiostrongyliasis (see Tharavanij, 1979); however, the best immunodiagnostic antigens have yet to be determined. A polypeptide with a specific molecular weight, prepared from the third-stage larvae, was found to stimulate a considerable amount of antibody (Dharmkrong-AT & Sirisinha, 1983). Thus antigens from larvae may be important in the immunodiagnosis of A. cantonensis, particularly in early infection. However, the incubation period-between infection and symptoms-of the disease in humans is more than 2 weeks (Hung & Chen, 1988). By this time, the larvae have developed to the juvenile stage in the CNS prior to the onset of illness. Some reports indicate that juvenile worm antigen gives better immunodiagnosis of angiostrongyliasis while other reports prefer adult worm antigens (Chen, 1986; Cross & Chi, 1982; Sato, Otsuru, Asato & Kinjo, 1977; Cross, 1978). Here the sensitivity and specificity in the detection of antibodies with adult worm antigen appeared very similar to those for young adult worm antigens. However, the sensitivity in the detection of IgG antibody in CSF specimens was significantly less than that in sera. Thus, adult and young adult worm antigens can be used in sero-diagnosis of A. cantonensis infection by ELISA; serum, rather than CSF, better illustrates the status of the infection in the patient. The immunoglobulins of the serum and the CSF of patients were compared qualitatively and quantitatively with those in fluids from the control subjects. Total IgG, IgA and IgM concentrations in the CSF of patients with eosinophilic meningitis were significantly greater than those in CSF from normal patients (Tungkanak et al., 1972; Chen, 1975). The total IgG concentration from infected patients was significantly higher than that in the normal controls only in one case (Tungkanak et al., 1972); however, significantly higher levels of IgE and IgM were observed in patients in another study (Chen, 1975). Perhaps the small sample size and the variety of patients were reasons for these divergent observations. The increase of any class of immunoglobulin in patients could not truly represent the total immune response which might include nonspecific components that would not react with worm
antigens. The serum IgG, IgA, IgM and IgE antibodies which act against both A. cantonensis adult and young adult worm antigens were elevated significantly in this present study. The increasing levels of the four classes of immunoglobulins in the CSF specific to worm antigens corresponded with changes in the immunoglobulins in the serum. The increase of specific immunoglobulins to A. cantonensis in the CSF may also come via the blood by changes in the permeability of the blood-brain barrier. However, some of the evidence indicates that local immunoglobulin synthesis is involved in the central nervous system in response to an infection with A. cantonensis (see Yoshimura & Soulsby, 1976). In the present study, the increasing rate of antigenspecific IgG antibody in patients was greater than that for IgM antibody seen 1 month after symptoms of A. cantonensis infection. This may be the reason why there was a significant rise in IgG antibody in our patients. Evidence from in vitro studies on IgG antibody suggested that antibody-dependent cellmediated cytotoxicity (ADCC) was involved (Yoshimura, Uchida, Sato & Oya, 1983; Yen, C. M., Unpublished PhD thesis, Kaohsiung Medical College, Taiwan, 1988) as the main protective mechanism against infections with A. cantonensis. Parasites or their eggs in the intestine of hosts can stimulate immune responses in the mucosa to produce IgA antibody which appears in the serum and as secretory IgA antibody in the intestinal lumen (David, 1982). Why IgA antibody levels were elevated significantly in the serum and CSF of patients with angiostrongyliasis remains obscure. It is well known that high serum levels of IgE have been observed in many helminth infections (Kojima, Yokogawa & Tada, 1972; Genta, Ottesen, Poindexter, Gam, Neva, Tanowitz & Wittner, 1983; Chen & Yen, 1984; Chen, 1975). IgE antibody can induce ADCC and damage schistosomula (Capron, Bazin, Joseph & Capron, 1981; Kojima, Niimura & Kanazawa, 1987). Significantly higher antigen-specific IgE antibody levels were observed here in patients with angiostrongyliasis. To demonstrate whether IgE antibody also has the ability of ADCC in infection with A. cantonensis requires a further study to be undertaken. Acknowledgemenrs-The authors wish to thank Kao-Pin Huang, Departments of Parasitology and Pediatrics, Kaohsiung Medical College, for assistance in collection of serum and CSF specimens. We thank Wun-Yuen Dow, Department of Parasitology, Kaohsiung Medical College, for carrying out the ELBA. REFERENCES CAPRON M.,
BAZIN H., JOSEPHM. & CAPRON A.
1981.
Evidence of IgE-dependent cytotoxicity by rat eosinophils. Journal of Immunology 126: 1764-l 769. CHEN E. R. 1979. Angiostrongyliasis and eosinophilic meningitis in Taiwan: a review. In: Studies on Angiostrongyliasis in Eastern Asia andAustralia (Edited by CROSS J. H.), 57-73. Special Publication of the U.S. Naval Medical Research Unit No. 2, Taipei, Taiwan.
Antibodies
in angiostrongyliasis
CHEN E. R. & YEN C. M. 1984. Study on the control of zoonotic clonorchiasis (III). Human survey, immunodiagnosis and treatment. National Science Council Monthly (R. 0. C.) 11: 1401-1408. CHEN S. N. 1975. Blood and cerebrospinal fluid findings in eosinophilic meningitis and antibody to Angiostrongylus cantonensis. Bulletin of Institute of Zoology, Academy Sinica 14: 109-l 13. CHEN S. N. 1986. Enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Angiostrongylus cantonensis. Transactions of the Royal Society of Tropical Medicine and Hygiene 80: 398-405. CHENGC. W., SATO Y. & CHEN E. R. 1984. Epidemiological and clinical observations on eosinophilic meningitis and meningoencephalitis of children caused by infection with A. cantonensis in southern Taiwan. Ryukyu Medical Journal 7: l-9. CHENG C. W., SATO Y., CHEN E. R. & OTSURU M. 1989. Application of enzyme-linked immunosorbent assay (ELISA) in serodiagnosis of angiostrongyliasis, with special reference to the reactivity of the extracts from adult and young adult worms. Chinese Journal of Parasitology 2: 3246. CROSS J. H. 1978. Clinical manifestation and laboratory diagnosis of eosinophilic meningitis syndrome associated with angiostrongyliasis. Southeast Asian Journal of Tropical Medicine and Public Health 9: 161-170. CROSS J. H. & CHI J. C. H. 1982. ELISA for the qletection of Angiostrongylus cantonensis antibodies in pstients with eosinophilic meningitis. Southeast Asian Journ@ of Tropical Medicine and Public Health 13: 73-76. DAVID J. R. 1982. Immune effector mechanisms against parasites. In: Immunology of Parasitic Infections (Edited by COHENS. &WARREN K. S.), pp. 74-98. Blackwell Scientific Publications, Oxford. DHARMKRONG-AT A. & SIR~SINHA S. 1983. Analysis of antigens from different developmental stages of Angiostrongylus cantonensis. Southeast Asian Journal of Tropical Medicine and Public Health 14: 154-l 62. GENTA R. M., O~ESEN E. A., POINDEXTERR., GAM A. A., NEVA F. A., TANOWITZH. B. & W~~TNERM. 1983. Specific allergic sensitization to Strongyloides antigens in human stro
