Detection of the circulating antigens CAA and CCA in human Schistosoma infections : immunodiagnostic and epidemiological applications

Detection of the circulating antigens CAA and CCA in human

Schistosoma infections : immunodiagnostic and

epidemiological applications

Lieshout, E.A. van

Citation

Lieshout, E. A. van. (1996, May 28). Detection of the circulating antigens CAA

and CCA in human Schistosoma infections : immunodiagnostic and

epidemiological applications. Retrieved from

https://hdl.handle.net/1887/17689

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Corrected Publisher’s Version

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Detection of the circulating antigens

CAA and CCA

in human Schistosoma infections:

immunodiagnostic and epidemiological

applications.

Detection of the circulating antigens

Detection of tiie circulating antigens

CAA and CCA in human Schistosoma infections:

immunodiagnostic and epidemiological applications.

PROEFSCHRIFT

ter verkrijging van de graad van Doctor aan de Rijksuniversiteit te Leiden,

op gezag van de Rector Magnificus Dr. L. L e e r t o u w e r , hoogleraar in de faculteit der Godgeleerdheid,

volgens besluit van het College v a n Dekanen te verdedigen op dinsdag 28 mei 1 9 9 6

te klokke 1 4 . 1 5 uur

door

Elisabeth A n t o i n e t t e van Lieshout

Promotiecommissie : Promotor: Referent: Overige leden: Prof. Dr. A . M . Deelder Prof. Dr. D.G. Colley

(University of Georgia, A t l a n t a , U.S.A.)

Prof. Dr. A . W . C . A . Cornelissen (Rijksuniversiteit Utrecht) Prof. Dr. B. Gryseels

(Instituut voor Tropische Geneeskunde, A n t w e r p e n , België)

Prof. Dr. J . C . van H o u w e l i n g e n Dr. A . M . Polderman

Prof. Dr. R.R.P. de Vries

T h e w o r k described in this thesis w a s performed at the Schistosomiasis D e p a r t m e n t (Division of Basic Research) of the Naval A m e r i c a n Medical Research U n i t - 3 ( N A M R U - 3 ) in Cairo, Egypt (Heads: Dr. M . M . M a n s o u r and Dr. D.A. Dean) and at the D e p a r t m e n t of Parasitology, University of Leiden, T h e Netherlands ( H e a d : Prof. Dr. A . M . Deelder).

Cover illustration: Egyptian hieroglyphic script depicting haematuria, p r o b a b l y due t o schistosomiasis h a e m a t o b i u m (based on the papyrus of K a h u n , 1 9 0 0 B.C.).

CIP-GEGEVENS KONINKLIJKE BIBLIOTHEEK, DEN HAAG

Lieshout, Lisette van

D e t e c t i o n of the circulating antigens C A A and C C A in h u m a n Schistosoma infections: immunodiagnostic and epidemiological applications / Lisette van Lieshout.

-[S.l. : s . n . ] . - III.

Proefschrift Rijksuniversiteit Leiden. M e t lit. o p g . -M e t s a m e n v a t t i n g in het Nederlands.

ISBN 9 0 - 9 0 0 9 2 6 3 - 3

Contents

1. Improved diagnostic performance of the circulating antigen assay in human schistosomiasis by parallel t e s t i n g for circulating anodic and cathodic antigens in

serum and urine. 2 7

2 . Evaluation of an ELISA for c o m b i n e d measurement of

C A A and CCA in schistosomiasis m a n s o n i . 3 7

3. A s s e s s m e n t of cure in schistosomiasis patients after c h e m o t h e r a p y w i t h praziquantel by q u a n t i t a t i o n of

circulating anodic antigen (CAA) in urine. 4 9

4 . Circulating cathodic antigen levels in serum and urine of schistosomiasis patients before and after c h e m o t h e ­

rapy w i t h praziquantel. 5 7

5. M o n i t o r i n g the efficacy of different doses of prazi­ quantel by quantification of circulating antigens in

6. Immunodiagnosis of schistosomiasis mansoni in a l o w endemic area in Surinam by d e t e r m i n a t i o n of the

circulating antigens C A A and CCA. 71

7. Detection of the circulating antigens C A A and CCA in serum and urine in a group of D u t c h travellers w i t h

acute schistosomiasis. 8 5

8. A simple t e c h n i q u e t o pretreat urine and serum sam­ ples for q u a n t i t a t i o n of schistosome circulating anodic

and cathodic antigen. 9 9

9. Analysis of w o r m burden variation in h u m a n Schisto­

soma mansoni infections by determination of serum

levels of circulating anodic antigen and circulating

cathodic antigen. 111

10. Comparison of circulating antigen levels in t w o areas endemic for schistosomiasis mansoni indicates local

List of abbreviations

A W A adult w o r m antigen

A W A - T C A trichloroacetic acid soluble f r a c t i o n of A W A BSA bovine serum albumin

9 5 % C I 9 5 % confidence interval C A A circulating anodic antigen CCA circulating cathodic antigen

ELISA enzyme-linked i m m u n o s o r b e n t assay epg eggs per gram of faeces

e / I O m I eggs per 10 ml of urine FITC fluorescein isothiocyanate IC i m m u n e complexes

IFA immunofluorescence assay

m g / k g milligram per kilogram (body weight) M A b monoclonal antibody

n number PBS phosphate-buffered saline SD Standard deviation

SEA soluble egg antigen

TR-IFMA time resolved i m m u n o f l u o r o m e t r i c assay v / v v o l u m e / v o l u m e

Preface

Schistosomiasis (bilharzia) is one of the major parasitic i n f e c t i o n s in t r o p i c a l areas. It is caused by blood-dwelling flukes, residing in t h e mesenteric and pelvic veins of the human host. Over 2 0 0 million individuals are estimated t o be infected w i t h these w o r m s , while at least 7 0 0 million people are at risk.

The c o n v e n t i o n a l m e t h o d t o diagnose this disease is by the d e m o n s t r a t i o n of parasite eggs in faecal or urine specimens. H o w e v e r , this t e c h n i q u e has several disadvantages, a.o. infections w i t h l o w w o r m burden can be easily m i s s e d , and due t o a high day-to-day f l u c t u a t i o n in egg c o u n t s , repeated e x a m i n a t i o n s are needed t o estimate the intensity of i n f e c t i o n . Alternatively, schistosomiasis can be diagnosed by the detection of host antibodies directed against s c h i s t o s o m e antigens. This t e c h n i q u e has s h o w n to be highly sensitive and specific, b u t is not able t o differentiate b e t w e e n active and past i n f e c t i o n , or t o give i n f o r m a ­ t i o n a b o u t intensity of i n f e c t i o n .

A comparatively n e w approach for the diagnosis of schistosomiasis is the d e m o n s t r a t i o n of Schistosoma antigens in the circulatory s y s t e m or the urine of the host. A l t h o u g h these so called "circulating antigens" may be p r o d u c e d by several parasite life cycle stages, m o s t research has been done on t w o g l y c o -c o n j u g a t e s w h i -c h are asso-ciated w i t h the gut of the adult w o r m , namely circulating anodic antigen (CAA) and circulating cathodic antigen (CCA).

The research described in this thesis should be considered as a c o n t i n u a t i o n of the initial w o r k p e r f o r m e d by De J o n g e et al.. It covers a period of seven years, of w h i c h a p p r o x i m a t e l y the first three years have been s p e n t at t h e N A M R U - 3 in Cairo, Egypt, the other four at the Department of Parasitology of the University of Leiden, The Netherlands. The main objectives of t h i s s t u d y w e r e t o further elaborate the assessment of C A A and CCA c o n c e n t r a t i o n s in h u m a n serum and urine samples, in particular for the diagnosis of an active

Schistosoma i n f e c t i o n and the assessment of cure f o l l o w i n g c h e m o t h e r a p y . In

a d d i t i o n , as serum and urine levels of C A A and CCA w e r e f o u n d t o be corre­ lated t o the number of w o r m s present, w e also evaluated t h e use of a n t i g e n d e t e r m i n a t i o n in more epidemiologically-oriented studies, t h a t is t o say t o examina i n f e c t i o n patterns and the d y n a m i c s of w o r m populations in h u m a n s living in endemic areas.

Chapter 1 of this thesis includes a general i n t r o d u c t i o n t o h u m a n s c h i s t o s o m i a s i s , w i t h particular emphasis on the diagnosis of t h i s parasitic disease. In a d d i t i o n , a literature o v e r v i e w is presented dealing w i t h t h e charac­ teristics of C A A and CCA, and w i t h the determination of C A A and CCA in serum and urine, b o t h in experimentally infected animals and in h u m a n s .

Chapter 2 describes the experimental w o r k , the majority of w h i c h has also appeared as papers in scientific journals. These publications are included as such w i t h o u t any m o d i f i c a t i o n s , w h i c h has resulted in some overlap, particularly in t h e " i n t r o d u c t i o n " and "material and m e t h o d " sections. F u r t h e r m o r e , some inconsistencies b e t w e e n the different papers w e r e unavoidable due t o t h e f a c t t h a t a f e w t e c h n i c a l aspects of the circulating antigen assays have c h a n g e d d u r i n g t h e years in w h i c h this w o r k w a s p e r f o r m e d .

chapter contains a further evaluation of the sensitivity and specificity of t h e C A A and CCA assays, both for serum and urine. The use of serum C A A and CCA d e t e r m i n a t i o n for the assessrhent of w o r m burdens in h u m a n s is reported in chapters 2 . 9 and 2 . 1 0 . A n t i g e n levels are analyzed in relation t o t h e age of the host, the parasite egg counts and the transmission level of the endemic area.

C h a p t e r 1

Human schistosomiasis

Schistosomes belong t o the class of Trematoda (Platyhelminthes). There are five s c h i s t o s o m e species w h i c h can be parasitic t o m a n : Schistosoma mansoni,

S. japonicum, S. haematobium, S. intercalatum and S. meliongi, of w h i c h t h e

first three are the m o s t c o m m o n . These species differ in a number of i m p o r t a n t w a y s , a.o. the species of the (intermediate) snail host, the final location in t h e (definitive) h u m a n host, the numbers of ova p r o d u c e d , the size and shape of t h e eggs and the p a t h o l o g y they induce.

Figure 1 s h o w s an adult male and female schistosome w o r m . The male is 1 t o 2 c m long and embraces the thinner and longer female in a g r o o v e on its ventral side called the gynaecophoric canal. The adult schistosome w o r m s have an average life span of 2 t o 5 years, but some may live for up t o 4 0 y e a r s .

.male

.female

Figure 1 . Paired adult Schistosoma w o r m s .

From G ö n n e r t , R. ( 1 9 4 8 ) . Die Struktur des Korperoberflache von Bilharzia

mansoni ( S a m b o n , 1 9 0 7 ) . Zeitschrift für Tropenmedizin und Parasitologie, 1 ,

adult worms

Biomphalaria sp.

Figure 2 . Life cycle of Schistosoma mansoni and Schistosoma haematobium. (Courtesy of Dr. A . M . Deelder)

The digestive s y s t e m comprises a short oesophagus and an intestine w h i c h divides into t w o latera! g u t caeca. The w o r m s feed on serum g l o b u l i n s , h a e m o g l o b i n and other c o m p o n e n t s of the host b l o o d . A s s c h i s t o s o m e s have no anus, undigested material, including parasite antigens, is e x c r e t e d by regular r e g u r g i t a t i n g .

Figure 2 illustrates the transmission cycle. A d u l t w o r m s do not m u l t i p l y in t h e h u m a n b o d y . The ova produced by the w o r m s need t o leave the b o d y t o c o n t i n u e the life cycle and transmission has to take place via the appropriate intermediate snail host. The number of eggs daily produced by a mature female w o r m varies f r o m several hundreds t o several t h o u s a n d s , d e p e n d i n g on t h e species. Histolytic e n z y m e s , secreted by the next larval stage called m i r a c i d i u m , enable t h e eggs t o penetrate t h r o u g h the host tissue to bladder (S. haema­

tobium) or intestine (other species), w h e r e t h e y are excreted w i t h t h e urine (S. haematobium) or faeces (other species). A b o u t half of the n u m b e r of e g g s ,

W h e n an excreted egg reaches fresh w a t e r , it hatches and the m i r a c i d i u m emerges. This larva m u s t , t o develop further, penetrate a compatible fresh w a t e r snail w i t h i n a period of several hours. The snails (Gastropoda) susceptible t o S.

mansoni belong t o the genus Biompfialaria, for S. haematobium and S. interca­ latum t o the genus of Bulinus, while S. japonicum and S. mekongi are

trans-mitted by the genus Oncomelania and Neotricula, respectively. In t h e snail, massive asexual multiplication occurs and several generations of m u l t i p l y i n g larvae (sporocysts) d e v e l o p . Eventually, these s p o r o c y s t s produce large n u m b e r s of infective larvae (cercariae), w h i c h leave the intermediate host in n u m b e r s up t o 3 0 0 0 cercariae per snail per day.

W h e n a h u m a n host is exposed t o infested w a t e r , cercariae are able t o penetrate the skin w i t h i n minutes. Via the blood s t r e a m , the y o u n g parasites (schistosomula) migrate, in a period of a f e w w e e k s , via the lungs t o the liver w h e r e the adult w o r m s pair. The paired w o r m s migrate t o the pelvic (S. haema­

tobium) or t h e mesenteric (other species) veins and a b o u t 4-7 w e e k s after

infection egg p r o d u c t i o n starts. The number of adult w o r m s present in an infected person is estimated t o range f r o m a couple of dozens, up t o several thousands.®

Occasionally, severe itching and dermatitis may develop shortly after cercarial penetration of the skin, particulary in those individuals w h o are exposed for the first t i m e . These s y m p t o m s ; h o w e v e r , are usually mild and o f t e n pass u n n o t i c e d . Severe dermatitis may also result f r o m invasion of n o n -human s c h i s t o s o m e cercariae, giving rise t o ' s w i m m e r s i t c h ' , w h i c h is c o m m o n in some non-tropical e n v i r o n m e n t s .

A c u t e schistosomiasis or 'Katayama fever' is associated w i t h t h e m a t u r a t i o n of the w o r m s and the onset of egg p r o d u c t i o n , and usually occurs 3 t o 9 w e e k s after i n f e c t i o n . S y m p t o m s are often non-specific, not related t o the intensity of i n f e c t i o n , and more o f t e n noticed in individuals infected for the first t i m e .

Schistosoma infection can be very difficult t o diagnose at this s t a g e , also

because it is usually to early to demonstrate eggs in the excreta.^®

epidemiological d a t a , schistosomiasis has also been associated w i t h the incidence of several t y p e s of cancer, of w h i c h the correlation b e t w e e n S.

haematobium i n f e c t i o n and cancer of the bladder seems t o be m o s t apparent.^'^°

The m o r t a l i t y rate due to schistosomiasis is not h i g h , and even serious hepatic or urinary m o r b i d i t y develops only in a small percentage of t h o s e i n f e c t e d . Still, high prevalences of less severe m o r b i d i t y (e.g. b l o o d y diarrhoea or haematuria) could have a significant impact on the general heaith s t a t u s of t h e involved c o m m u n i t i e s . ^ '

Intensity and d u r a t i o n of infection have been pointed o u t by m a n y investiga-tors as the major f a c t o r s determining d e v e i o p m e n t of serious chronic diseases in infected individuals, b u t has been disputed by o t h e r s . ' ^ Probably also other f a c t o r s (a.o. genetic and immunological background) play an i m p o r t a n t role. W h e n c o m p a r i n g different c o m m u n i t i e s , the relation b e t w e e n intensity of infec­ t i o n in a certain population and the prevalence of m o r b i d i t y seems t o be more p r o n o u n c e d . ' ^

The geographical distribution of schistosomiasis is b o t h linked t o t h e d i s t r i b u t i o n of the intermediate h o s t s , w h i c h breed only in tropical c l i m a t e s , and t h e lack of proper sanitation and w a t e r supply. A l t h o u g h e f f e c t i v e d r u g s are available, the number of areas w h e r e transmission is t a k i n g place is still increasing, due t o changing ecological and socio-geographical f a c t o r s (e.g. irrigation projects; human migration). Schistosomiasis is endemic in a p p r o x i m a t e l y 7 5 countries in the t r o p i c s , reaching f r o m China, via the M i d d l e East and A f r i c a , t o B r a z i l . ' ^ ' *

The dispersion of schistosomes in the h u m a n host p o p u l a t i o n f o l l o w s a negative binomial d i s t r i b u t i o n , i.e. in endemic areas the majority of t h e parasites is present in a small f r a c t i o n of the infected individuals. In a d d i t i o n , there is a characteristic age-related prevalence and intensity of i n f e c t i o n , w i t h a p r o n o u n c e d peak in egg excretion during adolescence, w h i c h rapidly decreases in adults. Figure 3 s h o w s an example of such a t y p i c a l age-intensity c u r v e for a h u m a n p o p u l a t i o n living in an area w i t h high S. m a n s o n / t r a n s m i s s i o n .

f a c t s provide a justification for vaccine d e v e i o p m e n t research.

Of the several available schistosomicides, praziquantel is n o w a d a y s considered as the drug of choice. It is m o s t w i d e l y used as either Biltricide®, p r o d u c e d by Bayer Leverkusen, Germany, or Distocide®, p r o d u c e d by Shin Poong Pharmaceutical Co. L t d , Korea. The Standard advised dosage is a single oral dose of 4 0 m g per kilogram of b o d y w e i g h t (kgbw) for all species, e x c e p t for

S. japonicum and S. melcongi, w h e r e the current regimes are either three doses

each of 2 0 m g / k g b w or t w o doses each of 3 0 m g / k g b w , all given in one d a y .

p o s i t i v e - • > 4 0 0 epg —^e— > 1 0 0 0 epg

v> « c <D ü i— (D CL I 0 0 n 8 0 6 0 4 0 2 0 -1 0 2 0 3 0 4 0 a g e ( y e a r s ) of t h e h u m a n h o s t 5 0

Figure 3 . Example of an age-prevalence curve for a human population living in an area w i t h intensive S. manso/?/transmission (n = 1 1 8 5 ) . epg = Schistosoma eggs per gram faeces. Data f r o m T s h a m a k a , M a n i e m a , Zaire, w i t h kind permission of Dr. A . M . Polderman.

Diagnosis of schistosomiasis

diagnostic assays can also serve as a research t o o i , for example in epidemiological studies, or for the evaluation of possible v a c c i n e c a n d i d a t e s . This central role of diagnosis has been recently e x t e n s i v e l y r e v i e w e d by

Feldmeier and Poggensee.'^

The ideal diagnostic procedure should have b o t h a high specificity and sensitivity, so it could be used t o diagnose light infections and t o assess failure of t r e a t m e n t . It should be kept in mind t h a t the predictive value of a diagnostic t e s t is n o t only related to specificity and sensitivity of the assay c o n c e r n e d , b u t is also depending on the prevalence of infection in the studied p o p u l a t i o n .

Ideally, the diagnostic m e t h o d of choice provides b o t h qualitative and q u a n t i t a t i v e i n f o r m a t i o n , as the d e v e i o p m e n t of disease m a y be linked w i t h intensity of i n f e c t i o n . In a d d i t i o n , the ideal test should be able t o diagnose the disease early after e x p o s u r e . Furthermore, the procedure should be easy t o p e r f o r m , also under field c o n d i t i o n s , and preferably w i t h o u t t h e need of skilled personnel or expensive e q u i p m e n t . A l s o , t e s t results should be reproducible and simple t o interpret.

Until n o w no single diagnostic test for schistosomiasis is available w h i c h fulfils all these requirements and the m e t h o d s w h i c h are available range largely in their characteristics. Therefore, the decision w h i c h of the c u r r e n t m e t h o d s t o use depends on several f a c t o r s , a.o. the logistic situatiën (e.g. w h e t h e r it is applied for a detailed s t u d y performed in a w e l l equipped laboratory or for the clinical diagnosis of individuals performed under field conditions) and t h e t y p e of i n f o r m a t i o n needed (e.g. the presence of an active i n f e c t i o n , the state of i n f e c t i o n , t h e p a t h o l o g y induced).

The c u r r e n t l y available procedures for the diagnosis of schistosomiasis can be generally divided in three different approaches: (i) direct parasitological m e t h o d s , (ii) indirect m e t h o d s and (iii) immunological m e t h o d s , w h i c h can be either direct or indirect.

Parasitological methods

The m o s t w i d e l y used diagnostic m e t h o d is the direct d e m o n s t r a t i o n of parasite eggs after microscopie examination of stool or urine. Schistosoma eggs are not d i f f i c u l t t o identify. They are 7 0 t o 1 8 0 yum long and feature a t y p i c a l terminal (S. haematobium, S. intercalatum) or lateral (other species) spine.

In the other Schistosoma species, eggs can be d e m o n s t r a t e d in a direct faecal smear. M o s t w i d e l y used in epidemiological settings is the Kato-Katz m e t h o d , in w h i c h a Standard a m o u n t (generally a volume equivalent t o 2 5 m g of faeces) is first sieved t o remove coarse particles and t h e n cleared by t h e addition of g l y c e r i n . A l t h o u g h this technique is based on v o l u m e and n o t on w e i g h t , results are usually expressed as eggs per gram faeces (epg). C o n c e n t r a t i o n t e c h n i q u e s , such as the formol-ether or glycerine s e d i m e n t a t i o n m e t h o d , usually increase the sensitivity, but give only s e m i q u a n t i t a t i v e i n f o r m a t i o n and are more difficult t o perform.^°

Hatching m e t h o d s can be used for qualitative diagnosis and t o check the viability of eggs. The detection of viable eggs may be of i m p o r t a n c e for the assessment of cure, as dead eggs can be present in the excreta for several w e e k s after c h e m o t h e r a p y , particularly in 5. haematobium i n f e c t i o n s . A l t h o u g h sensitive, this t e c h n i q u e can have variable test results. Examination of rectal biopsy specimens are also used for the qualitative diagnosis of Schistosoma i n f e c t i o n s . This is, h o w e v e r , an invasive technique not acceptable for routine screening.

In principle, parasitological m e t h o d s have a specificity of 1 0 0 % , a l t h o u g h in practica mistakes may be made. The sensitivity, h o w e v e r , can be l o w , particularly in S. mansoni infections w h e r e the a m o u n t of examined faeces in a direct faecal smear is relatively small in relation to egg e x c r e t i o n . In S.

japonicum infection egg p r o d u c t i o n is m u c h higher, but there the d i s t r i b u t i o n of

the eggs in the stool can be very irregular. So in either case, light i n f e c t i o n s can be easily missed. Besides, a strong day-to-day f l u c t u a t i o n has been d e s c r i b e d . In order t o piek up light infections and to obtain a more accurate measure of the intensity of i n f e c t i o n , repeated examinations are necessary.^''^* Even so, in v e r y light infections, the number of excreted eggs is o f t e n so small t h a t even after repeated microscopical e x a m i n a t i o n , the diagnosis cannot be confirmed.^'^'^^ The s h o r t c o m i n g s of the parasitological methods will be more extensively discussed in chapter 3 (general discussion) of this thesis.

Indirect methods

Schistosomiasis can also be diagnosed by indirect m e t h o d s , based on the use of clinical, biochemical or immunological disease markers. These t e c h n i q u e s are more related w i t h the presence of pathology t h a n w i t h the actual i n f e c t i o n and therefore will only be very briefly discussed in this paragraph. For a more detailed r e v i e w , see Feldmeier and Poggensee.'^

Only in chronic urinary schistosomiasis the clinical signs of i n f e c t i o n are more specific. The d i s t r i b u t i o n of questionnaires w a s f o u n d t o be useful for t h e Identification of areas of high risk for urinary schistosomiasis.^® Also h a e m a t u r i a , proteinuria and dysuria seem t o be useful indicators for the presence of a chronic S. haematobium i n f e c t i o n . A dip-stick m e t h o d t o d e t e r m i n e haematuria has been s h o w n t o be a sensitive t e c h n i q u e , suitable for mass s c r e e n i n g , a l t h o u g h w i t h a l o w specificity.^® Recent studies indicated eosinophil cationic protein (ECP) in urine as a potential marker for S. haematobium induced m o r b i d i t y . " ' ^ ^

Due t o its t e c h n i c a l i m p r o v e m e n t and field-applicability, u l t r a s o n o g r a p h y is increasingly u s e d , b o t h in hospitals and at the level of field s t u d i e s , a l l o w i n g visualization of t h e pathological a n a t o m i c a l changes caused by t h e Schistosoma i n f e c t i o n .

Immunological methods

I m m u n o d i a g n o s t i c t e c h n i q u e s can be generally divided in three c a t e g o r i e s : a n t i b o d y d e t e c t i o n , measurement of circulating antigens, and other t e c h n i q u e s , e.g. t h e celluier i m m u n e response t o certain schistosome antigens. Here, only the first t w o will be d i s c u s s e d . A list of review papers, some of w h i c h c o n t a i n a detailed description of the different immunological t e c h n i q u e s , is given at the end of t h i s chapter.

A w i d e range of tests is described for the d e m o n s t r a t i o n of specific antibodies, including different assay f o r m a t s , the use of c r u d e , purified or r e c o m b i n a n t antigens and the d e t e c t i o n of specific antibody i s o t y p e s . A l t h o u g h m o s t of these t e c h n i q u e s have s h o w n t o be highly sensitive and specific, their value is considerably lowered by the f a c t t h a t antibody levels generally do not differentiate b e t w e e n present and past infection and do n o t give any i n f o r m a t i o n about the intensity of i n f e c t i o n . The use of purified antigens, such as for example the egg antigen CEF6, seems t o give s o m e w h a t better results.^° H o w e v e r , the preparation of purified antigens still demands large quantities of parasite material. Until n o w no generally applied antigen has b e c o m e available via r e c o m b i n a n t DNA t e c h n o l o g y .

Table 1 . Characteristics of CAA and CCA.'

Characteristics Reference

C A A :

- circulating anodic antigen

- also k n o w n as SCA,GASP and GASCAP - negatively charged

- the c a r b o h y d r a t e chains consist of multiple disaccharide units containing A/-acetyl-galactosamine and glucuronic acid

3 2 3 3 - 3 5 3 6

3 7

CCA:

- circulating cathodic antigen - also k n o w n as ' a n t i g e n M '

- neutral or slightly positively charged

- the c a r b o h y d r a t e chains consist of multiple trisaccharide units (lewis x) containing f u c o s e , galactose

and A/-acetyl-galactosamine

- contains some epitopes crossreactive w i t h Schistosoma eggs

3 2 3 8 , 3 9 3 2

4 0 4 1

Both antigens are: - genus specific - TCA soluble - heat resistent

- sensitive t o periodate

- p r o d u c e d by the gut epithelium of the adult w o r m - also present in the primordial g u t cells

of cercariae and y o u n g w o r m s - d e m o n s t r a t e d in the h o s t ' s :

serum and urine milk (CCA only)

kidney, liver and spleen

- e x t r e m e l y stable, as illustrated by the d e t e c t i o n of C A A in m u m m y tissue 3 9 , 4 2 3 2 , 3 9 , 4 2 3 2 , 3 6 , 3 9 3 2 , 3 9 , 4 2 4 3 - 4 6 4 5 - 4 7 3 2 , 3 6 , 3 8 , 4 8 4 9 4 4 , 5 0 - 5 2 5 3 , 5 4

A d d i t i o n a l l y , specific antibody profiles are also of interest for research purposes, t o s t u d y specific i m m u n e responses. Immuno-epidemiological studies revealed an association b e t w e e n the levels of specific IgE and resistance against r e i n f e c t i o n , f o l l o w i n g c h e m o t h e r a p y . On the other hand susceptibility seems t o be associated w i t h an lgG4 response.®®®''

Living parasites excrete and secrete a number of different antigens into t h e circulation of t h e host. These antigens can be classified, a c c o r d i n g t o t h e life-stage of the parasite, into cercarial antigens,®®®^ adult w o r m associated antigens (e.g. tegument,®°®' or gut-associated®^), and egg antigens (mainly released as h a t c h i n g fluid®'®®).

The d e t e c t i o n of these antigens in the circulation of the host potentially offers a large number of diagnostic, epidemiological and research applications: a.o. the q u a n t i t a t i o n of w o r m b u r d e n , assessment of c h e m o t h e r a p e u t i c e f f i c a c y and the i m p a c t of c o n t r o l measurements. Furthermore, circulating antigen d e t e c t i o n could provide, in addition to egg c o u n t s , c o m p l e m e n t a r y i n f o r m a t i o n on the d y n a m i c s of parasite populations. It c o u l d , for e x a m p l e , be used for t h e evaluation of f u t u r e vaccine candidates, especially if these vaccines also have an impact on t h e egg p r o d u c t i o n of the parasites, w h i c h w o u l d make parasito­ logical diagnosis unreliable.

Several studies have s h o w n t h a t the major circulating antigens belong t o the g r o u p of the adult w o r m gut-associated circulating antigens. These antigens are released in t h e circulation of the host by regular regurgitating of the u n d i g e s t e d c o n t e n t s of t h e parasites g u t . M o s t research has c o n c e n t r a t e d on t w o g l y c o -c o n j u g a t e s : -cir-culating anodi-c antigen (CAA) and -cir-culating -cathodi-c antigen (CCA).®^®® Both antigens are named after their electrophoretic mobility at neutral pH. The characteristics of C A A and CCA have recently been e x t e n s i v e l y r e v i e w e d by Van Dam.®® Table 1 s h o w s a s u m m a r y of their m o s t i m p o r t a n t properties.

C A A w a s first described by Berggren and Weller, and has been further characterised by Gold et al., Nash et al., and Deelder et a / . .3 2 3 4 , 3 6 , 4 2 , 4 4 , 4 8 ^ ^^gs

recently f o u n d by Bergwerff et al. t h a t the major c a r b o h y d r a t e chains of t h i s p r o t e o g l y c a n have a novel polysaccharide s t r u c t u r e . ' ^

CCA, or ' a n t i g e n M ' , w a s independently f o u n d by Carlier et al. and Deelder

Table 2 . Review of data on the demonstration of CAA and CCA in animals, experimentally infected with Schistosoma species which are infectious to humans." A n i m a l / Schistosoma sp. A cercs antigen B f r o m w e e k 0 w o r m load D t r e a t m e n t Ref. IVIouse/ S. mansoni 2 5 - 2 0 0 C C A n.d. yes 1 w e e k 6 9 S. mansoni 2 5 - 2 5 0 C C A 5 yes n.d. 7 0 S. mansoni 6 0 - 1 2 0 C A A 1 yes 1 w e e k 7131 S. mansoni 1 0 - 1 0 0 C A A 3 yes n.d. 7 2 S. mansoni 1 0 0 C A A 5 no n.d. 7 3 S. mansoni 1 0 0 C C A n.d. no n.d. 7 3 S. mansoni 6 0 0 C A A 4 n.d. n.d. 41 S. mansoni 1 0 0 0 C C A IVi no n.d. 7 4 S. mansoni 1 0 0 0 C A A 2V2 no n.d. 7 4 S. japonicum 2 5 C C A n.d. yes n.d. 7 3 S. japonicum 2 5 C A A 2 yes^' n.d. 7 3 S. japonicum 5 - 2 8 C A A 4 yes n.d. 7 2 S. h a e m a t o b i u m 5 0 - 5 0 0 C A A 5 yes n.d. 7 2 S. intercalatum 2 5 - 7 5 C A A 5 yes n.d. 7 2 B a b o o n / S. mansoni 1 5 0 0 C A A 5 no n.d. 4 1 S. mansoni 1 5 0 0 C C A 5 no n.d. 41 S. h a e m a t o b i u m 1 5 0 0 C A A 6 n.d. n.d. 41

A b b r e v i a t i o n s : s p . = s p e c i e s ; A = number of cercariae; B = detectable, in w e e k s after infection; C = correlation w i t h w o r m load; D = start decrease, in w e e k s after t r e a t m e n t w i t h c h e m o t h e r a p y ; Ref. = reference number; n.d. = not done.

" Part of this table has been reproduced from V a n Dam ef a / . . ' " To obtain comparable sensitivity and specificity, this table only summarizes data on the detection of C A A and C C A by monoclonal antibody based assays.

^' This study contrasts w i t h others w i t h respect to t h e kinetic of C A A detectability; it seems uniikely t h a t C A A is already detectable 1 w e e k after infection w i t h 1 2 0 cercariae and reaches m a x i m u m level already 2 w e e k s after infection, w h e n t h e w o r m s are still very small and t h e gut not y e t fully d e v e l o p e d .

V a n Lieshout et al., unpublished d a t a .

The primary f u n c t i o n of both antigens is not exactly l<nown. Based on their highly g l y c o s y l a t e d s t r u c t u r e , t h e y probably play a role in the p r o t e c t i o n of t h e s c h i s t o s o m e g u t . * ' Recently, it has been argued t h a t t h e y may also p r o t e c t t h e w o r m against celluier immune effector mechanisms of the host, as C A A interacts w i t h the first c o m p l e m e n t c o m p o n e n t Clq,^® and CCA p r e s u m a b l y evokes an a u t o i m m u n e response due t o similarity w i t h a major g r a n u l o c y t e surface antigen.^®

Several t e c h n i q u e s for the d e m o n s t r a t i o n of these circulating antigens have been r e v i e w e d by De J o n g e . I n i t i a l l y , these assays w e r e based on t h e use of polyvalent antisera. The d e v e i o p m e n t of monoclonal a n t i b o d y (MAb) based s a n d w i c h ELISA's has made it possible t o d e t e c t C A A and CCA in a sensitive and highly specific menner.'®-'^ These M A b ' s have also been applied in some other t e c h n i q u e s , e . g . for a more field applicable d e m o n s t r a t i o n of these antigens.®°®*

Table 2 summarizes the available data on the d e t e c t i o n of C A A and CCA in experimental animal models. Generally, these antigens can be d e m o n s t r a t e d in the circulation approximately 3 t o 5 w e e k s after i n f e c t i o n , depending on the intensity of i n f e c t i o n . A significant correlation b e t w e e n antigen level and t h e number of adult w o r m s after perfusion, w a s f o u n d in all studies i n f e c t i n g w i t h different doses of cercariae. The relation b e t w e e n serum C A A level and w o r m burden w a s f o u n d t o be linear and not affected by suppression of t h e celluier i m m u n e s y s t e m . " Following successful c h e m o t h e r a p y , antigen levels decrease rapidly.

Initially, h u m a n studies w e r e mainly f o c u s e d on the d e t e c t i o n of C A A in serum for t h e diagnosis of schistosomiasis m a n s o n i . H o w e v e r , in later studies b o t h C A A and CCA w e r e d e m o n s t r a t e d in either serum or urine and in individuals i n f e c t e d w i t h other Schistosoma species, w i t h t h e e x c e p t i o n of S.

mel(ongi, w h i c h has not been examined so far. Table 3 summarizes t h e c u r r e n t

C A A C C A

Schistosoma species: serum urine serum urine S. mansoni d e t e c t e d ' ' 7 8 , 8 1 , 8 5 - 9 0 8 8 , 8 9 , 9 1 7 9 , 8 5 , 8 6 , 8 9 , 9 2 - 9 4 7 9 , 8 5 , 8 9 , 9 1 , 9 2 correlation"' 7 8 , 8 1 8 5 - 9 0 8 9 , 9 1 = ' 8 5 , 8 6 , 8 9 , 9 2 - 9 4 8 5 , 8 9 , 9 1 , 9 2 S. haematobium detected 9 5 9 1 , 9 5 9 5 9 5 correlation 9 5 9 5 9 5 S. intercalatum detected correlation 9 6 , 9 7 9 6 , 9 7 9 7 9 7 9 7 9 7 S. japonicum detected correlation 9 8 , 9 9 9 8 9 9 Specific studies: Prevalence and intensity in an endemic area 1 0 0 - 1 0 2 1 0 0 1 0 0 1 0 0 , 1 0 2 Follow up after c h e m o t h e r a p y 8 8 , 9 0 , 9 5 , 9 8 , 1 0 1 , 1 0 3 - 1 0 6 8 8 , 9 5 , 1 0 4 9 3 - 9 5 , 1 0 4 , 1 0 7 9 5 , 1 0 4 , 1 0 7 A c u t e infections/Individuals f r o m non-endemic area's 1 0 6 , 1 0 8 , 1 0 9 1 0 9 1 0 9 1 0 9 M e a s u r e m e n t of w o r m burden 1 1 0 , 1 1 1 1 1 0 , 1 1 1

" To obtain comparable sensitivity and specificity, this table only summarizes data on t h e detection of C A A and C C A by monoclonal antibody based assays. For a review of antigen detection in h u m a n s including assays based on polyvalent anti-sera, see De J o n g e . "

^' This thesis includes t h e references: 8 8 , 8 9 , 1 0 0 , 1 0 4 , 1 0 7 , 1 0 9 - 1 1 1 , '' Reference number.

"' Studies w h i c h f o u n d a significant correlation b e t w e e n antigen levels and egg c o u n t s .

=' Reference 8 6 A correlation b e t w e e n antigen level and epg w a s only found for cases w i t h intestinal schistosomiasis, not for cases w i t h the hepatosplenic f o r m of t h e disease.

The main objectives of the research described in t h i s thesis c o n c e r n t h e f o l l o w i n g three aspects of the determination of C A A and CCA in h u m a n s e r u m and urine samples: (i) t o investigate the application of these antigen assays for the diagnosis of an active Schistosoma i n f e c t i o n , (ii) t o further evaluate t h e use of these antigen assays for the assessment of cure f o l l o w i n g c h e m o t h e r a p y , and (iii) t o use these antigen assays for more epidemiologically oriented studies, i.e. for the assessment of w o r m burdens t o study i n f e c t i o n patterns in endemic areas.

Further reading and recent reviews:

General

- Jordan P, W e b b e G , Sturrock RF. H u m a n schistosomiasis. C A B International, Walling-f o r d , ( 1 9 9 3 ) .

- Olds G R . Progress and future initiatives in schistosomiasis. Current Opinion in Infecti­

ous Diseases ( 1 9 9 3 ) , 6 : 3 4 2 - 3 4 8 .

Control and morbidity

- Gryseels B. Morbidity and morbidity control of schistosomiasis mansoni in sub-saharan A f r i c a . T h e s i s , Leiden, ( 1 9 9 0 ) . - Gryseels B. Morbidity due to infection w i t h

Schistosoma mansoni: an u p d a t e . Tropical and Geographical Medicine ( 1 9 9 2 ) , 4 4 : 1 8 9

-2 0 0 .

- W H O Expert C o m m i t t e e . T h e control of schistosomiasis. World Health Organisation

Technical Report Series ( 1 9 9 3 ) , 8 3 0 : 1 - 8 6 .

Immunity and vaccine deveiopment

- B u t t e r w o r t h A E , Dunne D W , Fulford A J C , Thorne K J I , Gachuhi K, G u m a J H , Sturrock RF. H u m a n i m m u n i t y to Schistosoma man­

soni: observations on m e c h a n i s m s , and

im-plications for control. Immunological

Inves-tigations ( 1 9 9 2 ) , 2 1 : 3 9 1 - 4 0 7 .

- Capron A . I m m u n i t y to schistosomes.

Current Opinion in Immunology ( 1 9 9 2 ) , 4 :

4 1 9 - 4 2 4 .

- Capron A , Riveau G , Grzych J M , Boulan-ger D, Capron M , Pierce R J . D e v e i o p m e n t of

a vaccine strategy against h u m a n and bovine schistosomiasis. Background and u p d a t e . Tropical and Geographical Medicine ( 1 9 9 4 ) , 4 6 : 2 4 2 - 2 4 6 .

- Gryseels B. H u m a n resistance to Schisto­

soma infections: A g e or experience? Parasi­ tology Today ( 1 9 9 4 ) , 1 0 : 3 8 0 - 3 8 4 .

- Bergquist NR. Controlling schistosomiasis by vaccination: A realistic option? Parasito­

logy Today ( 1 9 9 5 ) , 1 1 : 1 9 1 - 1 9 4 .

Chemotherapy

- Shekhar K C . Schistosomiasis drug t h e r a p y and t r e a t m e n t considerations. Drugs ( 1 9 9 1 ) , 4 2 : 3 7 9 - 4 0 5 .

- Kumar V , Gryseels B. Use of praziquantel against schistosomiasis: A review of current status. International Journal of Antimicrobial

Agents ( 1 9 9 4 ) , 4 : 3 1 3 - 3 2 0 .

- Cioli D, Pica-Mattoccia L, Archer S. Antischistosomal drugs: past, present ... and future? Pharmacology and Therapeutics ( 1 9 9 5 ) , 6 8 : 3 5 - 8 5 .

Diagnosis and circulating antigens

- Qian ZL, W e n H C . Schistosome circulating antigens (CSA) as a possible diagnostic parameter for active infections. Acta

Leiden-sia ( 1 9 8 3 ) , 5 1 : 3 7 - 5 2 .

and Infectious Diseases ( 1 9 8 7 ) , 7 : 9 3 - 1 0 5 .

- De Jonge N. Immunodiagnosis of

Scfiisto-soma infections by detection of the circu­

lating anodic antigen. Thesis, Leiden, ( 1 9 9 0 ) .

- M a d d i s o n SE. Serodiagnosis of Parasitic Diseases. Clinical Microbiology Reviews ( 1 9 9 1 ) , 4 5 7 - 4 6 9 .

- Bergquist NR. Present Aspects of I m m u n o d i a g n o s i s of S c h i s t o s o m i a s i s .

Memorias Do Instituto Oswaldo Cruz

( 1 9 9 2 ) , 8 7 : 2 9 - 3 8 .

- Bergquist NR. Immunodiagnostic approa­ ches in schistosomiasis. J o h n Wiley & Sons L t d . , W e s t Sussex, ( 1 9 9 2 ) .

- Feldmeier H, Poggensee G . Diagnostic techniques in schistosomiasis control. A review. Acta Tropica ( 1 9 9 3 ) , 5 2 : 2 0 5 - 2 2 0 . - Deelder A M , Qian ZL, Kremsner P G , A c o s ­ ta L, Rabello A L T , Enyong P, Simarro PP, V a n Etten E C M , Krijger F W , Rotmans J P , Fillié Y E , De Jonge N, A g n e w A M , V a n Lies­ hout L. Quantitative diagnosis of Schisto­

soma infections by m e a s u r e m e n t of circula­

ting antigens in serum and urine. Tropical

and Geographical Medicine ( 1 9 9 4 ) , 4 6 : 2 3 3

-2 3 8 .

- V a n D a m G J . Circulating gut-associated antigens of Schistosoma mansoni biological, immunological, and molecular a s p e c t s . Thesis, Leiden, ( 1 9 9 5 ) .

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