Malarial antibodies in tropical splenomegaly syndrome in Papua New Guinea

308 TRANSA~IONS OF THE ROYAL SOCXETYOF TROPICAL MEDICME AND HYGIENE, VOL. 71, No. 4,1977.

Malarial antibodies in tropical splenomegaly Papua New Guinea 6. G. CkwwG*,

syndrome in

ANGELA GARDNER~,P. HUDSON%, BRYDGETHUDSON~ AND A. VOUXR~&, ‘Papua New Guinea Institute of Medical Research 2.Wufield Institute of Comparative Medicine, London 8London School @-Hygiene and Tropical Medicine

~a~a~~ae were estimated by indirect immL~nofluorescence in sera of New Guineans with TSS, and were compared with those ‘of normal controls sharing the same environment.

P.

Levels of species and class-specific malarial antibody were studied in 249 New Guineans with tropical splenomegaly syndrome (TSS) and in 87 control subjects living in the same area. Titres of IgG and IgM antibody to Plasmodiam falciparam, P. V&.X and P. rna~ar~ae were estimated by indirect immunofluorescence. Both IgG and IgM antibody levels were higher in subjects with TSS than in controls; IgM titres were highest in those with the greatest splenic enlargement. Responses to all three species were comparable. It is conchrcled that there is no evidence from this study to incriminate any one species of malaria parasite in the production of tropical splenomegaly syndrome. It is well established that elevated serum IgM concentrations (WELLS, 1968) and high malarial antibody titres (GEBBIEet al., 1964) are characteristic of tropical splenomegaly syndrome (TSS). IgM forms the major component of the ~oglobulins, believed to be ~mune complexes (ZIEGLER, 1973), which are found in the serum of over 80% of patients (WELLS, 1970). IgM has also been demonstrated within the Kupffer cells (ZIEGLER,1973), and on the surface of red cells (SSEBABIet al., 1975), and thus may play a key role in the production of many of the major manifestations of the disease (CRANE, 1977). In patients with gross splenomegaly it has been shown that the IgM includes malarial antibody to Plasmodium fakiparum (ZIEGLER et al., 1973). The extent to which IgM malarial antibody contributes to the general elevation in malarial antibody times and to the elevation in serum IgM levels in subjects with TSS has not been defined. The nature of the stimulus to the development of persistent splenomegaly and this overproduction of IgM remains unclear. Persistent infection with a particular species of Plasmodium has been considered likely on immunological and epidemiological grounds and P. vivax, P. malariae and P. falciparum have all come under suspicion, but no significant preponderance of infections by any one species in subjects with TSS has so far been demonstrated (MARSDEN & CRANE, 1976). The present study of TSS was undertaken to establish the nature of the elevation in malarial antibody titres in this disease, and to ascertain whether there was any serological evidence of excessive exposure or of an abnormal response to any one malarial species. Titres of IgG and IgM antibodies to P. falciparum, P. vivax and

Subjects and methods Subjects

The 249 subjects with splenomegaly came from four villages near the floor of the Upper Watut Valley of Papua New Guinea, at altitudes of 1,000 to 1,200 m above sea level, where malaria is meso~ndemic and transmission occurs throughout the year (CRANE & PRYOR, 1971). They represented some 80% of the population of these villages aged eight years or more. The diagnosis of TSS was made on the following grounds: in all cases splenomeg~y had persisted for some years, there was no clinical evidence of primary hepatic disease; in over 80% of subjects slow regression of splenomegaly subsequently occurred with long term antimalarial prophylaxis; and no other cause of persistent marked splenomegaly has been demonstrated in this extensively studied population (PRYOR, 1967). 35 of the control subjects came from the same villages and the other 52 from adjacent villages which are environmentally similar. All subjects belonged to the one linguistic group. None had received regular antimalarial therapy, althou~ all had access to an aid-post where chloroquine could be obtained for the treatment of acute febrile illnesses. The distribution of the subjects by age, sex and spleen grade is shown in Table I. At the time of examination the malaria parasite rate was 165 %, the species dis~ibution being P. vivax 53 %, P. falciparum 30 % and P. malariae 17 % of positive films. Sera

Blood was collected by venepuncture into plain sterile containers; serum was separated within 12 hours of collection without prior chilling, stored at -20” and transported to London in dry ice. At the time of collection the name, sex and apparent age of each subject was recorded. Age estimates were later checked against village birth records and revised where necessary. Spleen size was assessedby Hackett grade and also by measuring the maximum distance from left costal margin to the tip of the spleen. *Present address: Division of Haematology, Repatriation General Hospital, Concord, N.S.W. 2139, Australia.

309

G. G. CRANE et al.

Table I - Distribution of spleen grades by age and sex in the 336 subjects of the investigation.

Age

Group 5-9 10-14 15-19 20-29 30-39 4049 50-

0-1 0 2 2 14 17 3 4

Totals

42

Males Spleen Grade II-III IV-V 14 2 12 3 4 3 25 4 27 4 17 3 4 1 103

20

Females Spleen Grade O-I II-III IV-V 0 8 7 2 19 2 2 9 0 20 29 8 19 19 3 1 13 3 1 5 1 45

102

24

An figens

The antigens were obtained from experimentally infected laboratory monkeys-P. falciparum, P. vivax, P. malariae and P. brasilianum from Aotus frivirgatus, and P. cynomolgi from Macaca mulafa. Infected blood was considered suitable for use only if 10% or more of red cells were parasitized with predominantly late trophozoites or early schizonts, and if preliminary testing demonstrated acceptable sensitivity (i.e. if the preparation gave IgG malarial antibody titres > 1:1,280 with pooled hyperimmune Nigerian and New Guinean sera, and < 1 :lO with a negative control serum). A single batch of slides of each antigen was used throughout the investigation. Conjugates

Burroughs Wellcome monospecific fluoresceinconjugated antihuman IgG and anti-human IgM were used. Ampoules were reconstituted with distilled water and diluted with phosphate buffered saline, pH 7.2 (1:20 for anti-IgG, 1:6 for anti-IgM). After dilution, aliquots were stored at - 76” and were thawed once only, on the day of use. Control and standard sera

The positive control serum consisted of a pool of sera from 200 New Guinean adults with splenomegaly of varying degrees; the negative control serum was obtained from a healthy Caucasian who had never experienced a clinical attack of malaria. Human sera containing speciesspecific malarial antibodies were supplied as freeze-dried preparations by the Division of Biological Standards, Medical Research Council, and were reconstituted on the day of use in accordance with the directions. Malarial

antibody estimations

The indirect immunofluorescent technique of VOLLER & O’NEILL (1971) was employed. In all investigations sera were thawed and diluted, and the tests were completed on the one day. Antibody levels of New Guinean sera were determined by testing fourfold dilutions from 1:5 to 1:20,480 against falciparum, cynomolgi and brasilianum antigens. Intermediate values were interpolated where appropriate. Calculations

Negative results were arbitrarily assigned a titre of 2.5, the mean reading of the negative control serum. All calculations were performed after logarithmic transformation of individual values. Thus means which have been expressed as log2 values are geometric means.

The following preliminary investigations were performed in duplicate using doubling dilutions of sera, to establish the validity of the techniques: Vivax vs. cynomolgi and malariae vs. brasilianum antigens: titres of three MRC standard sera and 11 New Guinean sera to cynomolgiand vivax antigens were estimated, using IgG and IgM conjugates; a similar comparison of brasilianum and malariae antigens was performed in an identical manner. Comparable IgG and IgM titres to vivax and cynomolgi were obtained with all monospecific and hyperimmune sera. In no case was there more than a twofold difference in titres. Brasilianum antigen gave consistently higher titres than the malariae antigen employed in the test, indicating its greater sensitivity, but the specificities of the two antigens appeared identical. This established validity of employing cynomolgi antigen to estimate vivax titres, and brasilianum antigen in the measurement of malariae titres. Specificity

of the antigens

Cross-reactivity was assessed by estimating titres to falciparum, cynomolgi and brasilianum antigens in five MRC monospecific sera, using both conjugates. Only P. malariae antibody exhibited any significant crossreactivity; but, in all cases, titres against the homologous antigen were at least fourfold higher than against either other antigen, indicating adequate specificity of the test system. Rheumatoid factor

The possibility that rheumatoid factor was producing fallaciously high IgM titres was examined in a group of 14 New Guinean TSS sera. Rheumatoid factor was absorbed by three successive exposures to aggregated IgG by a modification of the method of TORRIGIAM & ROUT (1967). Pre- and post-treatment rheumatoid factor titres were estimated using Latex particles (Hyland Laboratories) sensitized with human SZfraction. IgG and IgM malarial antibody titres to P. falciparum were then estimated on pre- and post-absorption samples, on the day that the absorption was completed. The initial titres of rheumatoid factor in the 14 sera were 1:160 to > 1:640. After absorption, all but one titre was < 1:20. Absorption of rheumatoid factor did not alter IgG and IgM falciparum antibody titres in the majority of sera. In two cases only was there a fourfold decrease; this occurred with both IgG and IgM titres, and probably resulted from diluting errors rather than from removal of both IgG and IgM antibody. Thus IgM malarial antibody was shown to be distinct from rheumatoid factor. Results In the series as a whole there was significant correlation between all six titres. Thus it appeared that each subject tended to produce all antibodies equally readily or equally poorly. IgG titres to all antigens were consistently higher than IgM titres, and falciparum antibody levels were significantly higher than those to vivax and malariae (Table II). IgG antibody levels reached a peak in adolescence, whereas IgM antibody levels tended to rise progressively throughout life (Fig. 1). The correlation co-efficients between age and IgM antibody titres were significant for all three species. Relation

to spleen size

ZgG fitres to all species were significantly higher in subjects with TSS than in controls (Table II, Fig. 2).

310

MALARIAL ANTIBODIESIN TROPICALSPLENOMEGALYSYNDROMEIN PAPUA NEW GUINEA

Table II -Mean malarial antibody values for controls and adults with TSS. Parameter Controls TSS log2 malarial antibody titre IgG falciparum 10.490 11.759 malariae 7.743 9.736 vivax 8.236 9.077 IgM falciparum 4578 7.342 malariae 3.958 6.643 vivax 3.996 5.716 IgMjIgG ratio falciparum .409 .617 malariae *476 .679 vivax .453 .I24 Other ratios IgG mal/IgG falcip .731 .824 IgG vivax/IgG falcip .I93 *770 IgM mal/IgM falcip .941 .951 IaM vivax/IaM falciu .931 .912 Tit&s are expr&sed as log, values. All titres and IgM/IgG ratios were significantly greater in TSS than in controls: ratios of other titres to falciparum titres did not differ significantly between the two groups. However, mean levels were no higher in subjects with gross splenomegaly than in those with grade II or III spleens (Fig. 3). Thus the presence of splenomegaly, but not its degree, was related to IgG antibody levels. IgM titres showed a proportionately greater elevation in subjects with TSS than did IgG titres, and were higher still in those with gross splenomegaly. This was reflected in higher mean titres in subjects with grade IV and V spleens (Fig. 3) and in significant correlation co-efficients between IgM titres and spleen size (Table III). However, the degree of elevation in mean titre, and the correlation co-efficients, were comparable for IgM antibody to all three species. As is shown in Fig. 2, titres of < 1 :lO occurred only with IgM antibody; they were significantly more common in the control group (29%) than in subjects with TSS (4 %). Table III - Relationship between malarial antibody titres and spleen size, in all subjects with TSS. Correlation between spleen size and : Significance log IgG falciparum titre -.‘oll log IgG malariae titre -.023 :s log IgG vivax titre -.050 NS log IgM falciparum titre +.184 .Ol > P > .005 log IgM malariae titre +.169 .05>P> .Ol log IgM vivax titre + .265 .Ol > P > .005 r values are zero order correlation co-efficients. Spleen size was the distance between costal margin and lowest point of the spleen. ZgM/IgG antibody ratios As species differences in responses might be more apparent as alterations in the proportions of IgG and IgM antibody rather than as differences in titre, the ratio log IgM titre:log IgG titre was calculated for each species in each subject. In general these values merely reflected the behaviour of IgM antibodies: they rose progressively with increasing age, were higher in TSS than in controls (Table II), and in subjects with gross splenomegaly. They did not indicate particular involvement by any one species.

Vivaxjfalciparum and malariae/falciparum antibody ratios To compensate for possible differences in sensitivity of test systems involving different antigens, each vivax and malariae titre of both IgG and IgM antibody was calculated as a proportion of the corresponding falciparum titre for each patient. As is shown in Table II, this manoeuvre also failed to indicate differences in responses to the three species. Discussion The available evidence indicates that TSS arises in response to recurrent malarial infection, and to that alone (MARSDEN& CRANE,1976). METSELMR(1956) and PETERS& STANDFAST(1957) observed that splenomegaly was more common in adults in New Guinea, where P. vivax is the commonest species, than in Africa where P. falciparum predominates and P. vivax does not occur. It was suggested that the high adult snleen rates in New Guinea might result from intermitter , release of parasites from prolonged exo-erythrocytic c,cles of P. vivax or, less commonly, P. malariae. However, extensive field investigations in Papua New Guinea have not demonstrated an association between splenomegaly and parasitaemias of any one species, nor a predominance of any one species in areas where TSS is particularly common (MARSDENet al., 1967; CP.ANE& PRYOR,1971). In general, hospital studies in other countries have given similar results, subjects with TSS being noteworthy for the infrequency with which circulating malaria parasites of any species can be detected. These negative findings do not, however, necessarily disprove the hypothesis. Immunoglobulin studies in the Upper Watut Valley have indicated that the transition from simple malarious splenomegaly to TSS takes place as a gradual process between the ages of eight and 15 years (CRANEet al., 1971); and while there is no doubt that the subsequent perpetuation of the syndrome requires the presence of at least occasional circulating parasites (MARSDEN& CRANE, 1976), by that time the species causing the infection may well be irrelevant, for the critical events giving rise to development of the syndrome have already taken place, during or before the transitional phase of late childhood. If these events involve excessive exposure or an aberrant reaction to one particular species, they would be expected to leave behind some residual evidence, for example: (i) an elevation of specific IgG titre, indicating increased exposure in the past ; (ii) an elevation in specific IgM titre, indicative of the abnormal response; (iii) a deviation from the normal proportions of IgG and IgM antibody. The results of this investigation do not support any of these possibilities. Neither in adults, in whom the syndrome is established, nor in children, some at least of whom must be in the transitional phase, was there evidence of an atypical immune response to any one species. IgG falciparum titres were significantly higher than those of other species; but this was true both of subjects with TSS and of controls, and so reflects either enhanced immunogenicity of this parasite or a greater sensitivity of the falciparum test system. The elevation in IgM titres in subjects with TSS was comparable for all species. The greatest proportional elevation in those with gross splenomegaly occurred with IgM malariae and vivax antibodies, but correlation between titre and spleen size was greatest for falciparum and vivax antibody. In neither case was the difference between species responses of statistical significance. Thus

G. G. CRANE

311

et al.

IgG

antibody

IgM

antibody

4096

1024

256

64

I

1

10

20

I

years

30 of age

I

1

40

50

Fig. 1. Variation with age of IgG and IgM titres to P. falciparum (f) P. malariae (m), and P. vivax (v), in 288 subjects from four Watut villages. (The additional 52 subjects from other villages have been.excludedfrum.this analysis).

312

MALARIAL

ANTIBODIES

IN TROPICAL

SPLENOMEGALY

W

SYNDROME

IN PAPUA

NEW GUINEA

ANTIBODY

CONTROLS

I 25

falciparum

malariae

vivax

TSS

IgM

ANTIBODY

CONTROLS

TSS 50

25

0

r”l 2’

I’r i’I b-, I, 2’

falciparum

2’4

titre

2’

214

27

malariae

titre

J-

2’

vivax

2’

214 titre

Fig. 2. Frequency distribution of IgG and IgM antibody titres to the three speciesin controls and in adults with TSS.

G. G. CRANE etd.

falciparum

2

Q) z.w

12

313

I-1

IgG

antibody

v/1

IgM

antibody

malariae

vivax

ADULTS

-

28 -

24 -

i

l-1

CHILDREN 2

12

28 : E 24

1

I

0

dL ll-lll

IV-V

spleen

0

ll-lll

IV-V

0 grade

Fig. 3. Mean IgM and IgG antibody titres in adults and children, grouped according to spleen grade.

/ 3

314

MALARIAL

ANTIBODIEtS

IN TROPICAL

SPLENOMEGALY

there is no evidence to incriminate any one species of plasmodia aetiologically. In a small study of Ugandan patients, ZIEGLER et al. (1973) reported elevation of IgG and IgM titres to P. falciparum in subjects with gross splenomegaly. The present study has confirmed this observation. It has in addition demonstrated that this elevation in titres occurs in TSS with even minor splenomegaly, that it involves antibody directed towards all three species, and that the greatest proportionate increase is found in IgM titres (particularly where splenomegaly is gross). The high IgG titres in this disease do not necessarily imply a greater total exposure to malaria in the past. Although the outstanding immunological abnormality in TSS is the overproduction of IgM, a significant increase in IgG synthesis also occurs (CRANE et al., 1974). This increased production of IgG, much of which is malarial antibody (CRANE et al., in preparation), may also be a manifestation of the basic disturbance in the immune response to malaria which underlies the development of this disease. Acknowledgements The assistance of Dr. C. C. Draper of the London School of Hygiene and Tropical Medicine, and Dr. G. Huldt of National Bacteriological Laboratorv. Stockholm. in the provision of malarial-antigens; of Dr. P. Q’Neill of St. Thomas’ Hospital, London, in the rheumatoid factor study; and the financial support of the Wellcome Trust, is gratefully acknowledged. References Crane, G. G. (1977). The pathogenesis of tropical splenomegaly syndrome-the role of immune complexes. Papua New Guinea Medical Journal, in press. Crane, G. G., Pitney, W. R., Hobbs, J. R. & Gunn, C. (1971). Immunoglobulin levels in the Kaiapit and Upper Watut areas of New Guinea, with special reference to the tropical splenomegaly syndrome. Transactions of the Royal Society of Tropical Medicine and Hygiene, 65795-807.

Crane. G. G. & Prvor. D. S. (1971). Malaria and the tropical splenomegaly syndrome ’ in New Guinea. Transactions of the Royal Society of Tropical Medicine and Hygiene, 65, 3 15-323.

Crane, G. G., Wells, J. V. & Jones, B. P. (1974). The role of plasma proteins in chronic expansion of plasma volume in tropical splenomegaly syndrome. II. Metabolism of 1311-labelledIgG. Transactions of the Royal

SYNDROME

IN PAPUA

NEW GUINEA

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Gebbie, D. A. M., Hamilton, P. J. S., Hutt, M. S. R., Marsden, P. D., Voller, A. & Wilks, N. E. (1964). Malarial antibodies in idiopathic splenomegaly in Uganda. Lancet, ii, 392-393. Marsden, P. D., Connor, D. H., Voller, A., Kelly, A., Schofield, F. D. & Hutt, M. S. R. (1967). Splenomegaly in New Guinea. Bulletin of the World Health Organization, 36, 901-911. Marsden, P. D. & Crane, G. G. (1976). The tropical splenomegaly syndrome-a current appraisal. Revista do Instituto

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15, 635-649.

Peters, W. & Standfast, H. A. (1957). Report on a malaria survey in the Sepik District. Medical Journal of Australia, i, 861-868. Pryor, D. S. (1967). Tropical splenomegaly in New Guinea. Quarterly Journal of Medicine, 36, 321-336. Ssebabi, E. C. T., Jagwe, J. G. M., Nzaro, E. & Amsel, S. (1975). Tropical splenomegaly syndrome. An immune complex disease. East African Medical Journal, 52, 680-685. Torrigiani, G. & Roitt, I. M. (1967). Antiglobulin factors in sera from patients with rheumatoid arthritis and normal subjects. Quantitative estimation in different immunoglobulin classes.Annals of Rheumatic Diseases, 26, 334340. Voller, A. & O’Neill, P. (1971). Immunofluorescence method suitable for large scale application to malaria. Bulletin of the World Health Organization,

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Ziegler, J. L. (1973). Cryoglobulinaemia in tropical splenomegaly syndrome. Clinical and Experimental Immunology, 15, 65-78. Ziegler, J. L., Voller, A. & Ponnudurai, T. (1973). Malarial antibodies in tropical splenomegaly syndrome in Uganda. Tropical and Geographical Medicine, 25, 282-285. Accepted for publication

24th March,

1977.