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research on hookworms, using recently developed animal models 44 and by epidemiological projects linking immunological assays with resistance and worm loss in the field. Understanding the mechanisms involved in worm loss will be an important step in potential vaccine production, and I hope that a concerted effort to apply the tools of modern experimental medicine will help to elucidate aspects of hookworm biology which have eluded experimenters in the past. References 1 Banwell, J.G. and Schad, G.A (1978) Clin. Gastrc~ enterol. 7, 129-156 2 Foster, W.D. (1965) A History of Parasitology, E & S Livingstone Ltd, Edinburgh and London 3 Sturrock, R.F. (1967) E. Aft. Med.J. 44, 142-149 4 Miller, T.A. (1970) E. Aft. Med.J. 47, 334-363 5 Nwosu, A.B.C. and Anya, A.O. (1980) Tropenmed. Parasitol. 31,201-208 6 Goldsmid, ].M. (1965) Centr. Aft. J. Med. 11,160-167 7 Hoagland, K.E. and Schad, G.A. (1978) Exp. Parasitol. 44, 36-49 8 Hsieh, H.C. (1970)Jap.J. Parasitol. 19, 508-552 9 Knight, R. and Merren, T.G. (1981) Ann. Trop. Med. P arasitol. 75,299-314 10 Nawalinski, T. et al. (1978)Am. J. Trop. Med. Hyg. 27, 1152-1161 11 Udons~, J.K. (1984)Ann. Trop. Med. Parasitol. 78,443444 12 Cort, W.W. et al. (1923) Am. J. Hyg. 3, 85-110 13 Schad, G.A. et al. (1984) in Human Ecology and Infectious Diseases (Croll, N.A. and Cross, J., eds), pp. 188223, Academic Press, New York 14 Udonsi, J.K. et al. (1980)Zeitschr. Parasitenk. 63,251259 15 Maplestone, P.A. (1932) Ind. y. Med. Res. 19, 11451151 16 Nawalinski, T. et al. (1978) Am. J. Trop. Med. Hyg. 27, 1162-1173 17 SmiUie,W.G. (1922) Monogr. Rockefeller Inst., Second Paper 17, 1-73
18 Palmer, E.D. (1955)Am.J. Trop. Med. Hyg. 4, 756-757 19 Keymer, A.E. and Slater, A.F.G. (1987) Parasitol. Today 3, 56-58 20 Schad, G.A. et al. (1973) Science 180, 502-504 21 Nawalinski, T.A. and Schad, G.A. (1974)Am.J. Trop. Med. Hyg. 23,895-898 22 Maplestone, P.A. (1930) Ind.J. Med. Res. 18, 685-698 23 Schad, G.A. and Anderson, R.M. (1985) Science 228, 1537-1540 24 HasweU-Elkins, M.R. et al. Parasitology (in press) 25 Kendrick, J.F. (1934) Am. J. Trop. Med. Hyg. 15,363379 26 Miller, H.R.P. (1984) Vet. Immunol. Immunopathol. 6, 167-259 27 Stewart, D.F. (1955) Nature 176, 1273-1274 28 Dineen, J.K. et al. (1977) Int. J. Parasitol. 7, 211-215 29 Wakelin, D. (1985)Parasitol. Today 1, 17-23 30 Stewart, D.F. (1950)Austr.J. Agric. Res. 1,301-321 31 Behnke, J.M. (1987) inHuman Parasitic Diseases. Hookworms. (Gillies, H.M., ed.), Elsevier Science Publishers, Amsterdam (in press) 32 Hill, R.B. (1926) Am.J. Hyg. 6, 103-117 33 Sweet, W.C. (1925)CeyhmJ. Sci. Sect. D, Med. Sci. 1, 129-140 34 Seo, B.S. et al. (1980)KoreanJ. Parasitol. 18, 145-151 35 Kumar, N. et al. (1980) Ind. J. Med. Res. 71,531-537 36 Hotez, P. etal. (1985)J. Biol. Chem. 260, 7343-7348 37 Plaut, A.G. (1983)Ann. Rev. Microbiol. 37,603-622 38 Carroll, S.M. and Grove, D.I. (1985)Exp. Parasitol. 60, 263-269 39 Behnke, J.M. (1987) Adv. Parasitol. (in press) 40 Stokes, C.R. (1984)inLocallmmuneResponsesoftheGut (Newby, T.J. and Stokes, C.R., eds), pp. 97-141, CRC Press, Florida, USA 41 Kalkofen, U.P. (1974) Am. J. Trop. Med. Hyg. 23. 1046-1053 42 Klaver-Wesseling, J.C.M. et al. (1978) Zeitschr. Parasitenkd. 56, 147-157 43 McLaren, D.J. (1976)Adv. Parasiwl. 14, 195-265 44 Behnke, J.M. et al. (1986) Trans. R. Soc. Trop. Med. Hyg. 80, 146-149 45 Schad, G.A. et al. (1975) in Nuclear Techniques in Helminthology Research pp. 41-54, Int. Atomic Energy Agency, Vienna 46 Cart, A. and Pritchard, D.I. (1987) Mol. Biochem. Parasitol. 19, 231-258
Asymptomatic Malaria Infections- Do They Matter? B,M. Greenwood I
Medical Research Council
Laboratories, Fajara, Banjul, The Gambia
In non-immune patients, most P. falinfections result in high parasitaemias and severe symptoms unless treated early 1. Fortunately however, in areas where malaria is endemic and where most of the population have some immunity, this is not the case and severe, clinical infections are relatively uncommon. What proportion of new malaria infections causes symptoms in semi-immunes is uncertain. In The Gambia, we have found that children who live in a rural area experience about 1 clinical attack of malaria per year 2 and a similar incidence has been noted in Liberia 3. In our survey, children were visited only once a week so that mild attacks may have gone unrecorded between visits. But even in areas of high malaria transmis-
dparum
sion, it is unlikely that children experience more than 2 clinical attacks of malaria per year. In our study area in The Gambia, children are exposed to about 50 bites from infected mosquitoes per year (S. Lindsay et al. unpublished) but in other parts of Africa, where transmission is more intense, inoculation rates 2 or 3 times higher have been recorded 4. Not all sporozoite inoculations lead to infection but these entomological observations suggest that in endemic areas, only a small proportion of malaria infections reach a sufficiently high parasitaemia to cause an acute illness (Fig. 1). The remainder produce a low-level, asymptomatic parasitaemia which may last for weeks or months5. Are these asymptomatic infections of any clinical sig~)1987, Elsewer Publications, Cambridge 0169-4758/87/$02 00
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nificance? I believe that they are, and that they will become increasingly important as malaria vaccines come :into use. Low-level ParasitaemJias New World monkeys can be protected against blood-stage irdbctions with P. faldparurn by a variety of crude, partially purified and genetically-engineeredparasite peptides or proteins 6. In most experiments only partial protection, has been achieved despite the fact that powerful adjuvants, such as Freund's adjuvant, have been used in immunization. Thu,; it seems likely that the first generation of human blood-stage malaria vaccines, given with a less powerful adjuvant, will give only partial protection rather than sterile immunity. Sporozoite vaccines, although acting in a different way, may also give partial protection to semiimmunes 7. If sporozoite vaccines can reduce the level of hepatic infection, and consequently the initial level of red cell invasion, they may delay the development of a level of parasitaemia high enough to cause symptoms, 10ng enough for a semiimmune host to mount an effective immune response. Thus the main effect of the first generation of sporozoite and blood-stage vaccines may be to prevent high level parasitaemia rather than to prevent infection. A vaccine that could do this would have an enormous impact on mortality and serious morbidity from malaria. But vaccinated subjects might still experience repeated, low-level infections. To determine how important these might be we need to consider the late complications of malaria, and the conditions associated indirectly with malaria (Table 1), and to determine the relative importance of acute, clinical attacks and sub-clinical infections in their pathogenesis. This is not easy .to do but some clues are given by study of the effects of age on the incidence of indirect consequences of malaria, and by study of the outcome of malaria intervention campaigns.
Q 0:~ N
A. ACUTE CLINICAL MALARIA Treatment
C 0
0.0 o 3 . _C
Severe illness
~t
Clinical threshold
B. ASYMPTOMATIC MALARIA 0
'
o-5 I
Q_O
N¢
Partially effective immune response
Clinical threshold
Fig. I. A schematic comparison of the parasitological findings in acute and asymptomatic infections with P. falciparum.
severe, untreated P. faldparum infection. In others this is not the case and it has been suggested that their anaemia is brought about by means other than direct destruction of RBC. Several possible ways have been Table I. Late and indirect consequencesof malaria and their possible mechanisms
Condition Anaemia
Malaria species mainly P. falciparum
Mechanism red cell production 1' red cell destruction
Hyper-reactive malarial splenomegaly syndrome
P. falciparum and P. vivax
i' IgM and immune complex production
Nephrotic syndrome
P. malariae
immune complex deposition
Burkitt's lymphoma
P. falciparum
1' B-cell turnover T-cell control over EBV-infected B-cells
Bacterial infections
P. falciparum
Immunosuppression Haemolysis
Anaemia
P. falciparum can cause a severe, acute, haemolytic anaemia by direct destruction of red blood cells (RBC). "['his occurs most frequently in non-immunes in whom very high levels of parasitaemia may occur. In endemic areas, young ctfildren who present with severe anaemia and[ malaria sometimes have only a low level of parasitaemia. In many such cases a history of a recent, acute febrile illness can be obtained and their anaemia probably results from a previous,
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RETtCULO ENDOTHELIAL SYSTEM
/i:!77
::(:
Activated phagocytic cell
Fig. 2. Suggested mechanism of anaemia in persistent malaria infections, by suppression o fred blood cell production and increased destruction of RBC.
suggested by which low-level malaria parasitaemia could cause anaemia including impairment of RBC production and enhanced RBC destruction (Fig. 2). Disturbed function of the bone marrow (marrow dyserythropoiesis) has been described both in patients with acute, symptomatic malaria and in children with low-level malaria parasitaemiaS,9. Also, it has been suggested that repeated malaria infections can, by increasing red cell turnover, lead to deficiencies in haematinics in people on a marginally sufficient diet. For this reason some physicians recommend folate supplements for children with malaria-associated anaemia, but there is no evidence to support this practice. In fact, serum and red cell folate levels are higher in children with malaria than in controlsm,n perhaps because malaria parasites synthesise folates. As malaria causes predominantly a haemolytic anaemia it would not be expected to lead to iron deficiency. However, in areas where malaria is thought to be a major cause of anaemia in young children, anaemia is often hypochromic and microcytic - suggesting iron deficiency. Iron metabolism is disturbed during acute episodes of malaria 12 and the possible effects of recurrent, asymptomatic malaria infections on iron metabolism need further investigation. Studies with chromium-labelled RBC
have shown that splenic clearance of nonparasitized RBC is enhanced for some time after an episode of acute, symptomatic malarial,13 perhaps due to hypersplenism. Splenic enlargement is found frequently in healthy children in malarious areas and this might be associated with an enhanced rate of clearance of non-parasitised RBC. I am unaware of any study in which this has been specifically investigated, but there is little doubt that hypersplenism is an important cause of anaemia (and sometimes leucopenia and thrombocytopenia) in patients with the hyper-reactive malarial splenomegaly syndrome (see below). The idea that immunological factors play an important part in the pathogenesis of malarial anaemia was once fashionable but is now held less widely. Early studies showed that sera from subjects resident in malarious areas frequently contained heterophile antibodies, cold agglutinins, and antibodies that reacted with altered human RBC 14. However, there is no evidence that any of these antibodies are directly haemolytic in vivo. Several studies demonstrated positive direct anti-globulin tests (Coombs tests) in some patients with malaria. For example, a series of studies in The Gambia found positive Coombs tests in approximately 50% of healthy village children and in a similar proportion of patients with acute, clinical malaria 15-]7. But positive Coombs tests cor_
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related only weakly with the presence of parasitaemia or anaemia 15. Another study of Gambian patients with P. falciparum malaria found no correlation between anaemia and a positive antiglobulin test 18 and, in Thai patients with acute malaria, sensitisation of RBC could not be demonstrated 19. In Gambian children 15, positive antiglobulin tests were due most frequently to bound C3d. IgG was present less often 15. When present, bound IgG was usually of the IgG2 or IgG4 sub-classes but anaemia was related only to the ]presence of bound IgG117. Immunoglobulin eluted from IgGpositive RBC contained antibody to P. falciparum schizonts - compatible with the idea that erythrocyte sensitisation resulted from passive attachment to RBC of complement fining, malaria antigen-antibody complexes 16. Attempts have been made to quantify the amount of immunoglobulin present on the surface of RBC obtained from malaria patients with a positive anti-globulin test. Nigerian patients showed some correlation between the amount of bound IgG and the presence of anaemia 2° but this was not the case in The Gambia where low levels of IgG binding (90-897 molecules per red cell) were noted 21. Much higher levels of binding would probably be required to facilitate erythophagocytosis. Thus, it appears that in the majority of cases where a positive antiglobulin test is found in patients with malaria, or in children resident in malarious areas, this is of no clinical consequence. In malaria endemic areas, anaemia is found most frequently in younger children suggesting that acute malaria infections play an important part in its pathogenesis. This idea is supported by the iact that successful malaria control campaigns have had a greater effect on haemoglobin levels or packed cell volumes (PC,V) in young children than in older children or adults23, 24. However in Tanzania, malaria control did have a small but sign~icant effect on haemoglobin levels in adults 23, and in The Gambia, we have found considerably higher PCVs in children given malaria prophylaxis than in children given treatment for acute infections alone (B.M. Greenwood et al. unpublished). Thus it is probable that acute and sub-clinical infections both contribute to the anaemia found frequently in children in areas where malaria is endemic. Malaria and the Outcome of Pregnancy Immunity to malaria -- acquired gradually throughout childhoc~i - may be lost during pregnancy, especially in primag-
Table 2. Evidence implicating malaria in the pathogenesis of the hyper-reactive malaria splenomegaly syndrome (supporting refs cited in Ref. 14) •
The condition is found only in areas where malaria is endemic.
•
Possessionof the haemoglobin genotype AS provides protection against the syndrome.
•
Patients with HPIS have higher levels of malaria antibodies than controls.
•
Hypergammaglobulinaemia and splenomegaly regress in patients who take malaria chemoprophylaxis.
ravidae. In semi-immune women, malaria infections during pregnancy rarely cause an acute febrile illness of the type seen in children, but can contribute to the anaemia of pregnancy and, occasionally, cause a severe, fife-threatening haemolytic anaemia25. Low-level, asymptomatic infections in the mother can cause parasitisation of the placenta26. This is important because it leads to lowering of the birth weight which has an adverse effect on child survival.
Splenomegaly Acute, clinical episodes of malaria can cause splenomegaly which regresses after the infection has been treated or resolved. But when malaria infections are recurrent, splenomegaly does not regress between attacks and a high proportion of children resident in malaria-endemic areas have modestly enlarged spleens. Whether these children are disadvantaged in any way is In a few subjects, binding of uncertain. As discussed above, it is possible large amounts oflgG I to that splenomegaly causes enhanced RBC especiallyif combined destruction of RBC but whether this is clin- with C3, may lead to enhanced RBC destruction. ically important is unknown. Spleens However, a recent report enlarged as a result of infection with P. that human RBC coated vivax seem more prone to rupture than nor- with IgG immune complexes mal spleens but this does not seem to be the are not taken up in vitro by phagocytic cells22 makes it case for P. falciparum. uncertain how this is brought In malaria endemic areas the prevalence about. of splenomegaly declines as immunity to malaria is acquired and, in most communities, few adults have enlarged spleens. However, in a proportion of subjects repeated exposure to malaria results in massive enlargement of the spleen and overproduction of IgM and malaria antibodies. This condition - originally termed the tropical splenomegaly syndrome (TSS) - is now called the hyper-reactive malarial splenomegaly syndrome (HMS) 27 to differentiate it from other forms of massive splenomegaly of unknown cause28,29. HMS is found only in areas where malaria is endemic but the prevalence of the syndrome varies considerably from region to region. In some villages in Papua New Guinea and in Indonesia a high proportion of the adult population are affected30, yet the condition is generally rare
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Genetic factors are probably important in the development of riMS. In West Africa the syndrome is found more frequently amongst Fulas (Fulanis) than other ethnic groups2&31, In Papua New Guinea4s there is some association between HMS and presence of the HLA antigen DR2.
Fig. 3. A schematic representation of the possible pathogenesis of the hyper-reactive malarial splenomegaly syndrome.
in areas of West Africa with a similar malaria endemicity31. The predominant clinical feature of HMS is massive enlargement of the spleen which may cause local discomfort32. Some degree of anaemia is usually found which is due partly to hypersplenism and partly to hypervolaemia. Acute haemolytic crises may occur. Liver biopsy usually shows infiltration of hepatic sinusoids with lymphocytes but this is not an invariable finding nor is it specific for HMS. For reasons that are not understood, patients with HMS have a poor prognosis. Nearly 50% of a large group of patients studied in Papua New Guinea died during a 15 year follow up period 33, and a similar high mortality has been noted in Nigeria and The Gambia (Ref. 34 and B.M. Greenwood et al. unpublished). Patients with HMS have very high serum levels of IgM and immune complexes which possibly represent immunoglobulin aggregates rather than true antigen/antibody complexes35. Clearance of these complexes is probably the main reason for the massive splenomegaly36. Cell mediated immune responses are usually normal; infiltration of
hepatic sinusoids with T-lymphocytes may represent a cellular immune response to antigens present in Kuppfer cells37. The pathogenesis of HMS is still only partially understood (Fig. 3) but there is strong evidence (Table 2) that malaria has a dominant role. Malaria is a powerful stimulus to immunoglobulin production and, amongst normal subjects resident in malarious areas, serum levels of IgM and autoantibodies rise throughout life. It seems likely that suppressor mechanisms are normally activated to overcome this malariadriven immune hyper-reactivity, but these are probably defective in patients with HMS. Studies in Indonesia38 have shown that patients with HMS have reduced numbers of T8 (suppressor) cells and normal levels of T4 (helper) cells, giving a high T4/ T8 ratio. In vitro studies show that IgM production by B-cells obtained from patients with HMS can be suppressed by normal T8 cells38 - strongly suggesting that the abnormality in patients with HMS lies in their T8 lymphocyte sub-population. Sera of Indonesian patients with HMS contain an antibody that can lyse T8 cells in the presence of complement; this autoantibody39, perhaps induced by malaria, may be the cause of the poor T-cell suppressor function found in patients with HMS. Lack of control over B-cell proliferation is probably the basic immunological defect in HMS, but it is not certain that this is brought about in all patients in the same way. The prevalence of HMS differs between Indonesia and Papua New Guinea, and West Africa, and there are differences in the clinical features of the syndrome in these areas. West African patients with HMS sometimes have a lymphocytosis which may be marked enough to suggest a diagnosis of leukaemia4°, whereas this is rarely, if ever, encountered in Indonesian or Papuan patients. It is important that the elegant in vitro studies on immunoglobulin production undertaken in Indonesian patients with HMS should be repeated in Africa. There is clear evidence that repeated subclinical infections of malaria are more important than acute symptomatic infections in causing HMS. Thus, HMS is primarily a disease of adults rather than of children, and the clinical and immunological features of the syndrome recur rapidly in adults brought into remission with chemoprophylaxis who stop taking their treatment.
Nephrotic Syndrome The incidence of the nephrotic syndrome
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in children is much higher in some parts of the tropics than in countries with a temperate climate. In part, tiffs is because the syndrome can result from infection with P. malariae (Table 3). The malarial nephrotic syndrome is seen most frequently in children aged 5-10 years, but it also occurs in adults - especially in East Africa42. Renal biopsies from children with the malarial nephrotic syndrome in Nigeria, showed thickening of the capillary basement membrane with focal and segmental sclerosis of glomerular tufts. On electron microscopy, sub-endothelial deposits and lacunae within the basement membrane were seen43,44. The prognosis of children with the malarial nephrotic syndrome is poor and most progress to chronic renal, failure and death, despite treatment with corticosteroids or immunosuppressive agents. The P. malariae nephrotic syndrome is, at least in part, an immune-complex nephritis. Studies in Nigeria and Uganda have shown deposits of in~nunoglobulin and complement in the glomerular basement membrane of nearly all cases4~,46. The pattern of immunoglobulha staining seen on immunofluorescence varies from a nodular to a more linear pattern, and in about one third of cases, P. mala~iae antigen has been demonstrated within basement membrane deposits. Yet despite these characteristic immunological changes,the pathogenesis of quartan malaria nephropathy is not straightforward. Unlike most other forms of infection-related immune-complex nephritis, the condition does not resolve when malaria is treated and reinfection prevented by chemoprophylaxis. This suggests that renal damage may be sustained by autoimmune mechanisms but this possibility has not been investigated adequately. What proportion ofP. malariae infections leads to renal damage, and why this happens in some children but not in others, remain unknown. In semi-immunes, P'. malariae infections rarely cause acute clinical illness with high levels of parasitaemia. Most lead to asymptomatic infections with persistent, low-level parasitaemia. Thus it is likely that these asymptomatic infections, rather than acute clinical episodes, are the main cause of the P. malariae nephrotic syndrome. This concept is supported by data from the malaria eradication campaign in Guyana47. Before eradication, chronic nephritis accounted for 7.8% of deaths in children and 13.5% of deaths in adults, among whom acute clinical episodes of malaria would have been infrequent. Following
21 I
Table 3. Evidence implicating P. malariae in the pathogenesis of the nephrotic syndrome (supporting refs cited in Ref. 14) •
The age distribution of the nephrotic syndrome in West Africa parallels that of P. malariae infection.
P. malariae is found 5 times more frequently in African patients with the nephrotic syndrome, than in controls. In Africa, patients with the nephrotic syndrome have higher levels of antibody to P. malariae, than controls.
P. malariae antigens can be demonstrated in glomerular deposits in about one third of African patients with the nephrotic syndrome. The nephrotic syndrome can be induced experimentally by infecting monkeys with P. malariae. Eradication of malaria in Guyana led to a fall in the incidence of the nephrotic syndrome.
eradication, chronic nephritis ceased to be a cause of death in children and accounted for only 2.5% of deaths in adults.
Burkitt's Lymphoma Burkitt's lymphoma is one of the commonest causes of childhood cancer in tropical Africa and some other parts of the tropics. In these areas it occurs in approximately 1 per 10 000 children per year4s. The tumour is seen most frequently in 2-16 year old children, with a mean age at presentation of about 7 years. A tumour of the jaw is the most usual presenting feature but multiple tumours are common and may occur in the abdomen and at other sites. Histological examination shows a characteristic 'starry sky' appearance of macrophages against a background of undifferentiated, round lymphocytes. The malignant lymphocytes of Burkitt's lymphoma have B-cell markers 49 and the tumour is a monoclonal B-cell lymphoma. Spontaneous regression occasionally occurs and, except in the most advanced stages, the tumour responds well to chemotherapy. Cytogenetic studies have shown that all, or nearly all, Burkitt's tumours show chromosomal translocationsS0, 51. The corn-
Some authors believe that the histological changes associated with P. malanae nephrotic syndrome are su~ciently characteristic to justify the designation of 'quartan malaria nephropathy', In Uganda however, more variable histological changes have been reported.
Table 4. Evidence implicating infection with Epstein-Barr virus (EBV) in the pathogenesis of Burkitt's lymphoma EBV DNA is integrated into the nuclear DNA of nearly all African cases of Burkitt's lymphoma. •
Children who subsequently develop Burkitt's lymphoma have higher levels of antibody to EBV viral capsid antigen (VCA) than controls.
•
EBV can 'immortalize' B-cells in in vitro cultures.
•
EBV can induce lymphomas in tamarind monkeys.
•
TamarindscanbeprotectedagainsttheoncogeniceffectsofEBVinfectionby vaccination.
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Table 5. Evidence implicating malaria in the pathogenesis of Burkitt's lymphoma (supporting refs cited in Ref. 14) •
A high incidence of Burkitt's lymphoma is found only in areas where malaria is endemic.
•
Within malarious areasthe incidence of Burkitt's lymphoma is low in areaswhere malaria infection is naturally infrequent or has been controlled.
and differentiation52. Fusion of c-myc with an Ig heavy chain constant gene appears to cause loss of normal growth regulation and uncontrolled proliferation of B-lymphocytes.
The Epstein Barr virus (EBV) plays an important part in the aetiology of Burkitt's lymphoma (Table 4). However, EBV is not • The haemoglobin genotype AS is under-represented in patients with Burkitt's essential to the pathogenesis of Burkitt's lymphoma. tumour and histologically indistinguishable • Malaria infection of experimental animals can enhance the oncogenic potential of lymphomas have been described in subjects tumour viruses. with no evidence of EBV infection. Infection with EBV is universal but a high incimonest abnormalities are translocations dence of Burkitt's lymphoma is found only between part of the long arm of chromo- in tropical areas with a high prevalence of some 8 and chromosomes 2, 14 or 22 which malaria - suggesting that malaria plays are all loci ofimmunoglobulin genes. These some part in its pathogenesis (Table 5). rearrangements are almost exclusive to BThere are two main hypotheses for the cell lymphomas. The translocated part of role of malaria in the pathogenesis of Burchromosome 8 contains an oncogene (c-myc) kitt's lymphoma. One postulates that by which normally influences cellular growth stimulating immunoglobulin-producing cells, malaria increases the likelihood of a chromosomal translocation at the site of one of the genes controlling immunoglobulin production, thus causing the transfer of the c-myc oncogene to an area where it permits unrestrained growth of B-cells53. P. falciparum can cause immortalization of Blymphocytes in culture but it is not known whether these transformed cells show chromosomal translocations 54. The primary event could be activation of B-cells by malaria leading to chromosomal translocation, followed by EBV infection and consequent immortalization of the transformed cells52, or it could be primary EBV infection and immortalization of B-ceUs subsequently stimulated by repeated malaria infections and thus increasing the likelihood of chromosomal translocation53 (Fig. 4). A second hypothesis suggests that malaria impairs the host's ability to control the growth of EBV-infected immortalized B-cells once these have been formed. In The Gambia it was shown that the ability of Tlymphocytes to control the growth of EBVinfected B-cells was impaired in children with acute malaria, but control was regained on recovery55. This loss of suppressor activity was associated with a change in the T4/T8 lymphocyte ratio. In Papua New Guinea, lymphocytes from subFig. 4. Two possible mechanisms for the interaction of malaria and Epstein Barr Virus (EBV) in the pathogenesis of Burkitt' s lymphoma. The lymphoma seems to be caused by jects resident in malarious areas were less uncontrolled growth of B-cells infected with EBV, which show a chromosomal transloca- effective at controlling the growth of EBVtion affecting cellular growth and differentiation. The inducer-suppressor T-cell chain is infected B-cells in vitro, compared to lyminhibited by malaria, allowing the tumour to develop. phocytes from residents of non-malarious Malaria also acts in the initiation of the B-cell transformation. In hypothesis A, the areas 56. B-cells are first immortalized by infection with EBV, and their multiplication is then The relative contributions of acute and stimulated by repeated malaria infections - thus increasing the likelihood of sub-clinical malaria infections to the chromosomal translocation. In hypothesis B, the primary event would be malaria infection promoting B-cell multiplication and chromosomal translocation, followed by pathogenesis of Burkitt's lymphoma is difEBV infection which immortalizes the transformed cells. ficult to assess. The age distribution of the
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tumour - maximum in children in the age range at which severe clinical infections are frequent - suggests that: these may be the most important factor but interpretation is complicated by the role of EBV infection which is also age-related. And if loss of Tcell control of EBV-infected B-cells is an important pathogenic factor, then sub-clinical infections could be important. The studies in Papua New Guinea which showed impaired T-cell control over EBVinfected B-cells were canied out in adults in whom acute episodes of malaria would have been unlikely. Immunomodulation Malaria is both immunostimulatory and immunosuppressive57. In endemic areas, healthy individuals have high serum IgG and IgM, and raised titres of rheumatoid factor and other autoantibodies 14. How malaria causes polyclonal B-cell activation is not fully understood, but repeated exposure to parasites with variant antigens and the production of mitogens by the parasites, are probably both involved. Yet despite immunological over-reactivity, some immune functions are suppressed in patients with malaria. Antibody responses to a variety of antigens are impaired - particularly to poorly immunogenic antigens such as capsular polysaccharides58. Do these immunological changes have any clinical importance? There is evidence in experimental animals that malaria increases susceptibility to some other infections and we have shoran recently that P. falciparum malaria predisposes to Salmonella infections in Gambian children 59. It is also possible that the immunomodulation produced by malaria could influence susceptibility to immune-mediated noninfectious diseases but this is difficult to investigate. Some evidence to support the idea that malaria has art indirect effect on mortality from other conditions is given by intervention studies. In 3 successful malaria control schemes in Africa, infant and child mortality were reduced by a greater extent than predicted from previous death rates attributed to malaria 6°. At Pare-Taveta in Tanzania and Kisumu in Kenya - both areas of high malaria endemicity - successful control of malaria with insecticides led to a reduction of mortality in adults as well as in children, suggesting that in these communities sub-clinical :malaria infections contributed in some indirect way to adult mortalitytl,62. This idea is also supported by results from Guyana where mortality from pneumonia, as well as from nephritis, fell in
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adults as well as in children as malaria came under control47.
Long-term Consequences of Malaria Consideration of the long-term consequences of malaria and the part played by subclinical infections allows us to make some guesses about what might happen in a community protected from acute, clinical malaria by a partially effective vaccine. My guess is that the prevalence of Burkitt's lymphoma and - if a partially protective P. malariae vaccine was introduced - of the malaria nephrotic syndrome, would decline but that neither condition would disappear. Some improvement in the overall haemoglobin level of the population might be anticipated. I would be surprised if a partially protective vaccine had much effect on the incidence of the hyper-reactive malaria splenomegaly syndrome, but in most malaria endemic areas, Burkitt's lymphoma, quartan malaria nephropathy and HMS are relatively infrequent complications of malaria. Persistence of these conditions might be a low price to pay for the booster immunizing effect of asymptomatic malaria infections in subjects given a malaria vaccine that was only partially protective. References
1 Phillips, R.E. and Warrell, D.A. (1986) Parasitol. Today 2,271-282 2 Greenwood, B.M. et al. (1987) Trans. R. Soc. Trop. Med. Hyg. (in press) 3 Bj6rkman, A. et al. (1986) Ann. Trop. Med. Parasitol. 80, 155-167 4 Druihle, P. et al. (1986) Infect. Immunity 53, 393-397 5 GlUes, H.M. (1986) Trans. R. Soc. Trop. Med. Hyg. 80, 353-359 6 Anders, R.F. (1985)Parasitol. Today 1,152-155 7 Nussenzweig, R.S. and Nussenzweig, V. (1985) Parasitol. Today 1,150-152 8 Phillips, R.E. etal. (1986) Quart.J. Med. 277, 305-323 9 Abdalla, S. etal. (1980)Brit.J. Haematol. 46, 171-183 10 Bradley-Moore, A.M. et al. (1985) Ann. Trop. Med. Parasitol. 79, 585-595 11 Oppenheimer, S.J. and Cashin, P. (1986) Trans. R. Soc. Trop. Med. Hyg. 80, 169-170 12 Srichaikul, T. etal. (1969)Arch. Intern. Med. 124, 623628 13 Woodnfff, A.W., Ansdell, V.E. and Pettitt, L.E. (1979) Lancet i, 1055-1057 14 Greenwood, B.M. and Whittle, H.C. (1981) Immunology of Medicinc in the Tropics. Edward Arnold, London 15 Facer. C.A., Bray, R.S. and Brown, J. (1979) Clin. Exp. Immunol. 35, 119-127 16 Facer, C.A. (1980) Clin. Exp. lmmunol. 39, 279-288 17 Facer, C.A. (1980) Clin. Exp. Immunol. 41, 81-90 18 AbdaUa, S.H. and Weatherall, D.J. (1982) Brit. J. Haematol. 51,415-425 19 Merry, A.H. etal. (1986)Brit.J. Haematol. 64, 187-194 20 Jeje, O.M., Kelton, J.G. and Blajchman, M.A. (1983) Brit. J. Haematol. 54, 567-572 21 Abdalla, S.H. (1986) Brit. J. Haematol. 62, 13-19 22 Sherwood, T.A. and VireUa, G. (1986) Clin. Exp. lmmunol. 64, 195-204 23 Draper, C.C. (1960) Brit. Med.J. i, 1480-1483 24 Delmont, J. et al. (1961)Bull. Soc. Path. Exot. 74,600610
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214 25 Fleming, A.F. (1981) Clin. Haematol. 10, 983-985 26 McGregor, I.A., Wilson, M.E. and Billewicz, W.Z. (1983) Tram. R. Soc. Trop. Med. Hyg. 77, 232-244 27 Bryceson, A.D.M. et al. (1983) Tram. R. Soc. Trop. Med. Hyg. 77,879 28 Bryceson, A.D.M., Fleming, A.F. and Edington, G.M. (1976) Acta Trop. 33, 185-214 29 De Cock, K.M. et al. (1983) Brit. Med. J. ii, 1347-1348 30 Crane, G.G. et al. (1985) Papua New Guinea Med. J. 28, 27-34 31 Greenwood, B.M. et al. (1987) Ann. Trop. Med. Parasitol. (in press) 32 Marsden, P.D. and Crane, G.G. (1976) Rev. Inst. Med., Sao Paulo 18, 54-70 33 Crane, G.G. (1986) Parasitol. Today 2, 4-9 34 Fakunle, Y.M. and Greenwood, B.M. (1980) Trans. R. Soc. Trop. Med. Hyg. 74, 419 35 Fakunle, Y.M. et al. (1978)Clin. Exp. Immunol. 31, 5558 36 Ziegler, J.L. (1973) Clin. Exp. Immunol. 15, 65-78 37 Fakunle, Y.M. and Greenwood, B.M. (1982) Clin. Exp. Immunol. 48, 546-550 38 Hoffman, S.L. etal. (1984) N. Engl.J. Med. 310, 337341 39 Piessens, W.F. et al. (1985) J. Clin. Invest. 75, 18211827 40 Fakunle, Y.M. etal. (1979) Trop. Geogr. Med. 31,353358 41 Bhatia, K.K. and Crane, G.G. (1985)Human Immunol. 13,235-242 42 Kibukamusoke, J.W. (1973) Nephrotic Syndrome of Quartan Malaria. Edward Arnold, London 43 Hendrickse, R.G. et al. (1972) Lancet i, 1143-1149
44 Abdurrahman, M.B. et al. (1983) E. Afr. Med. J. 60, 467-471 45 Allison, A.C. et al. (1969) Lancet i, 1232-1238 46 Ward, P.A. and Kibukamusoke, J.W. (1969) Lancet i, 283-285 47 Gigliogi, G. (1972)Bull. W H O 46, 181-202 48 Burkitt, D.P. (1970) in Burkitt's Lymphoma (Burkitt, D.P. and Wright, D.H., eds) pp. 186, Livingstone, London 49 Preud'homme, J.L. et al. (1985) in Burkitt's Lymphoma: a Human Cancer Model (Lenoir, G.M., O'Connor, G. and Olweny C.L.M., eds) pp. 47, IARC, Lyon 50 Manolov, G. and Manolova, Y. (1972) Nature 237, 3334 51 Zech, L. et al. (1976) Int. J. Cancer 17, 47-56 52 Lenoir, G.M. and Bornkamm, G.W. (1987) in Advances in Viral Oncology vol. 7 (Klein, G. ed.) pp. 173, Raven Press, New York. 53 Klein, G. (1979) Proc. NatIAcad. Sci. 76, 2442-2446 54 Kataaha, P.K. et al. (1984) Clin. Exp. Immunol. 56, 371-376 55 Whittle, H.C. et al. (1984) Nature 312,449-450 56 Moss, D.J. etal. (1983) Int.J. Cancer 31,727-732 57 Weidanz, W.P. (1982) Brit. Med. Bull. 38, 167-172 58 Williamson, W.A. and Greenwood, B.M. (1978) Lancet i, 1328-1329 59 Mabey, D.C.W., Brown, A. and Greenwood, B.M. (1987)J. Infect. Dis. 155, 1319-1321 60 Molineaux, L. (1985) in Health Policy, Social Policy and Mortality Prospects (Vallin, J. and Lopez, A. eds) pp. 13, Ordina Editions, Liege 61 Pringle, G. (1969) Tram. R. Soc. Trop. Med. Hyg. 63, $2-S18 62 Payne, D. et al. (1976)Bull. W H O 54,369-377
Antibacterial Action of Myiasiscausing Flies G.R, Erdmann Some species of calliphorid blowflies* lay their eggs in wounds; their larvae develop by feeding on the tissue, and the infection is known as myiasis or fly-strike. But wounds, from whatever cause, are frequently contaminated with bacteria - many of which can spread in the bloodstream causing septicaemia and~or toxaemia. For example, wound contamination with Clostridium welchii - leading to 'gas gangrene' - was a frequent cause of death amongst battlefield casualties. It is from such situations that early observations were made on the beneficial effect of sorne blow~y larvae in limiting the bacterial infection of wounds. Indeed, some military surgeons would deliberately infest wounds with blowfly maggots in order to prevent bacterial complications. Now, a century or two later, the search for new antibiotics had led researchers back to these early observations, and in this article, Gary Erdmann des- ~ ...... tribes progress in understanding the antibacterial action of blowfly maggots.
Battlefield casualties frequently died from septicaemia following bacterial infection of wounds. Surgeonsin Napoleon's Armies noted, however, that wounds infested with blowfly maggots often did not go septic ~ resulting in higher survivorship of wounded soldiers. ~ ~ ?
iii~ii
¸¸
A
One of the first observations of wound infestations was by D.J. Larrey, a military surgeon in Napoleon's army I. He recorded that larvae of a 'blue fly' common in S' (possibly Chrysomyia or Wohlfahrtia; see Refs 2-4) frequently invaded the wounds of soldiers while on thehe Alfi battlefield. Presence of the larvae helped / to remove scar tissue and reduced the threat of bacterial m infection. During the US civil war. J.F.
[]
*Formerly, the family Calliphoridae included the subfamilies Calliphorinaeand Sacrophaginae; species of both groups lay their eggs in damaged, decayingor dead verLebrate1issues.More recently, both subfamilieshavebeenelevatedto familyrank. ~)1987, Elsevier Publications,Cambridge 0169-4758/87/$0200
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research on hookworms, using recently developed animal models 44 and by epidemiological projects linking immunological assays with resistance and worm loss in the field. Understanding the mechanisms involved in worm loss will be an important step in potential vaccine production, and I hope that a concerted effort to apply the tools of modern experimental medicine will help to elucidate aspects of hookworm biology which have eluded experimenters in the past. References 1 Banwell, J.G. and Schad, G.A (1978) Clin. Gastrc~ enterol. 7, 129-156 2 Foster, W.D. (1965) A History of Parasitology, E & S Livingstone Ltd, Edinburgh and London 3 Sturrock, R.F. (1967) E. Aft. Med.J. 44, 142-149 4 Miller, T.A. (1970) E. Aft. Med.J. 47, 334-363 5 Nwosu, A.B.C. and Anya, A.O. (1980) Tropenmed. Parasitol. 31,201-208 6 Goldsmid, ].M. (1965) Centr. Aft. J. Med. 11,160-167 7 Hoagland, K.E. and Schad, G.A. (1978) Exp. Parasitol. 44, 36-49 8 Hsieh, H.C. (1970)Jap.J. Parasitol. 19, 508-552 9 Knight, R. and Merren, T.G. (1981) Ann. Trop. Med. P arasitol. 75,299-314 10 Nawalinski, T. et al. (1978)Am. J. Trop. Med. Hyg. 27, 1152-1161 11 Udons~, J.K. (1984)Ann. Trop. Med. Parasitol. 78,443444 12 Cort, W.W. et al. (1923) Am. J. Hyg. 3, 85-110 13 Schad, G.A. et al. (1984) in Human Ecology and Infectious Diseases (Croll, N.A. and Cross, J., eds), pp. 188223, Academic Press, New York 14 Udonsi, J.K. et al. (1980)Zeitschr. Parasitenk. 63,251259 15 Maplestone, P.A. (1932) Ind. y. Med. Res. 19, 11451151 16 Nawalinski, T. et al. (1978) Am. J. Trop. Med. Hyg. 27, 1162-1173 17 SmiUie,W.G. (1922) Monogr. Rockefeller Inst., Second Paper 17, 1-73
18 Palmer, E.D. (1955)Am.J. Trop. Med. Hyg. 4, 756-757 19 Keymer, A.E. and Slater, A.F.G. (1987) Parasitol. Today 3, 56-58 20 Schad, G.A. et al. (1973) Science 180, 502-504 21 Nawalinski, T.A. and Schad, G.A. (1974)Am.J. Trop. Med. Hyg. 23,895-898 22 Maplestone, P.A. (1930) Ind.J. Med. Res. 18, 685-698 23 Schad, G.A. and Anderson, R.M. (1985) Science 228, 1537-1540 24 HasweU-Elkins, M.R. et al. Parasitology (in press) 25 Kendrick, J.F. (1934) Am. J. Trop. Med. Hyg. 15,363379 26 Miller, H.R.P. (1984) Vet. Immunol. Immunopathol. 6, 167-259 27 Stewart, D.F. (1955) Nature 176, 1273-1274 28 Dineen, J.K. et al. (1977) Int. J. Parasitol. 7, 211-215 29 Wakelin, D. (1985)Parasitol. Today 1, 17-23 30 Stewart, D.F. (1950)Austr.J. Agric. Res. 1,301-321 31 Behnke, J.M. (1987) inHuman Parasitic Diseases. Hookworms. (Gillies, H.M., ed.), Elsevier Science Publishers, Amsterdam (in press) 32 Hill, R.B. (1926) Am.J. Hyg. 6, 103-117 33 Sweet, W.C. (1925)CeyhmJ. Sci. Sect. D, Med. Sci. 1, 129-140 34 Seo, B.S. et al. (1980)KoreanJ. Parasitol. 18, 145-151 35 Kumar, N. et al. (1980) Ind. J. Med. Res. 71,531-537 36 Hotez, P. etal. (1985)J. Biol. Chem. 260, 7343-7348 37 Plaut, A.G. (1983)Ann. Rev. Microbiol. 37,603-622 38 Carroll, S.M. and Grove, D.I. (1985)Exp. Parasitol. 60, 263-269 39 Behnke, J.M. (1987) Adv. Parasitol. (in press) 40 Stokes, C.R. (1984)inLocallmmuneResponsesoftheGut (Newby, T.J. and Stokes, C.R., eds), pp. 97-141, CRC Press, Florida, USA 41 Kalkofen, U.P. (1974) Am. J. Trop. Med. Hyg. 23. 1046-1053 42 Klaver-Wesseling, J.C.M. et al. (1978) Zeitschr. Parasitenkd. 56, 147-157 43 McLaren, D.J. (1976)Adv. Parasiwl. 14, 195-265 44 Behnke, J.M. et al. (1986) Trans. R. Soc. Trop. Med. Hyg. 80, 146-149 45 Schad, G.A. et al. (1975) in Nuclear Techniques in Helminthology Research pp. 41-54, Int. Atomic Energy Agency, Vienna 46 Cart, A. and Pritchard, D.I. (1987) Mol. Biochem. Parasitol. 19, 231-258
Asymptomatic Malaria Infections- Do They Matter? B,M. Greenwood I
Medical Research Council
Laboratories, Fajara, Banjul, The Gambia
In non-immune patients, most P. falinfections result in high parasitaemias and severe symptoms unless treated early 1. Fortunately however, in areas where malaria is endemic and where most of the population have some immunity, this is not the case and severe, clinical infections are relatively uncommon. What proportion of new malaria infections causes symptoms in semi-immunes is uncertain. In The Gambia, we have found that children who live in a rural area experience about 1 clinical attack of malaria per year 2 and a similar incidence has been noted in Liberia 3. In our survey, children were visited only once a week so that mild attacks may have gone unrecorded between visits. But even in areas of high malaria transmis-
dparum
sion, it is unlikely that children experience more than 2 clinical attacks of malaria per year. In our study area in The Gambia, children are exposed to about 50 bites from infected mosquitoes per year (S. Lindsay et al. unpublished) but in other parts of Africa, where transmission is more intense, inoculation rates 2 or 3 times higher have been recorded 4. Not all sporozoite inoculations lead to infection but these entomological observations suggest that in endemic areas, only a small proportion of malaria infections reach a sufficiently high parasitaemia to cause an acute illness (Fig. 1). The remainder produce a low-level, asymptomatic parasitaemia which may last for weeks or months5. Are these asymptomatic infections of any clinical sig~)1987, Elsewer Publications, Cambridge 0169-4758/87/$02 00
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nificance? I believe that they are, and that they will become increasingly important as malaria vaccines come :into use. Low-level ParasitaemJias New World monkeys can be protected against blood-stage irdbctions with P. faldparurn by a variety of crude, partially purified and genetically-engineeredparasite peptides or proteins 6. In most experiments only partial protection, has been achieved despite the fact that powerful adjuvants, such as Freund's adjuvant, have been used in immunization. Thu,; it seems likely that the first generation of human blood-stage malaria vaccines, given with a less powerful adjuvant, will give only partial protection rather than sterile immunity. Sporozoite vaccines, although acting in a different way, may also give partial protection to semiimmunes 7. If sporozoite vaccines can reduce the level of hepatic infection, and consequently the initial level of red cell invasion, they may delay the development of a level of parasitaemia high enough to cause symptoms, 10ng enough for a semiimmune host to mount an effective immune response. Thus the main effect of the first generation of sporozoite and blood-stage vaccines may be to prevent high level parasitaemia rather than to prevent infection. A vaccine that could do this would have an enormous impact on mortality and serious morbidity from malaria. But vaccinated subjects might still experience repeated, low-level infections. To determine how important these might be we need to consider the late complications of malaria, and the conditions associated indirectly with malaria (Table 1), and to determine the relative importance of acute, clinical attacks and sub-clinical infections in their pathogenesis. This is not easy .to do but some clues are given by study of the effects of age on the incidence of indirect consequences of malaria, and by study of the outcome of malaria intervention campaigns.
Q 0:~ N
A. ACUTE CLINICAL MALARIA Treatment
C 0
0.0 o 3 . _C
Severe illness
~t
Clinical threshold
B. ASYMPTOMATIC MALARIA 0
'
o-5 I
Q_O
N¢
Partially effective immune response
Clinical threshold
Fig. I. A schematic comparison of the parasitological findings in acute and asymptomatic infections with P. falciparum.
severe, untreated P. faldparum infection. In others this is not the case and it has been suggested that their anaemia is brought about by means other than direct destruction of RBC. Several possible ways have been Table I. Late and indirect consequencesof malaria and their possible mechanisms
Condition Anaemia
Malaria species mainly P. falciparum
Mechanism red cell production 1' red cell destruction
Hyper-reactive malarial splenomegaly syndrome
P. falciparum and P. vivax
i' IgM and immune complex production
Nephrotic syndrome
P. malariae
immune complex deposition
Burkitt's lymphoma
P. falciparum
1' B-cell turnover T-cell control over EBV-infected B-cells
Bacterial infections
P. falciparum
Immunosuppression Haemolysis
Anaemia
P. falciparum can cause a severe, acute, haemolytic anaemia by direct destruction of red blood cells (RBC). "['his occurs most frequently in non-immunes in whom very high levels of parasitaemia may occur. In endemic areas, young ctfildren who present with severe anaemia and[ malaria sometimes have only a low level of parasitaemia. In many such cases a history of a recent, acute febrile illness can be obtained and their anaemia probably results from a previous,
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RETtCULO ENDOTHELIAL SYSTEM
/i:!77
::(:
Activated phagocytic cell
Fig. 2. Suggested mechanism of anaemia in persistent malaria infections, by suppression o fred blood cell production and increased destruction of RBC.
suggested by which low-level malaria parasitaemia could cause anaemia including impairment of RBC production and enhanced RBC destruction (Fig. 2). Disturbed function of the bone marrow (marrow dyserythropoiesis) has been described both in patients with acute, symptomatic malaria and in children with low-level malaria parasitaemiaS,9. Also, it has been suggested that repeated malaria infections can, by increasing red cell turnover, lead to deficiencies in haematinics in people on a marginally sufficient diet. For this reason some physicians recommend folate supplements for children with malaria-associated anaemia, but there is no evidence to support this practice. In fact, serum and red cell folate levels are higher in children with malaria than in controlsm,n perhaps because malaria parasites synthesise folates. As malaria causes predominantly a haemolytic anaemia it would not be expected to lead to iron deficiency. However, in areas where malaria is thought to be a major cause of anaemia in young children, anaemia is often hypochromic and microcytic - suggesting iron deficiency. Iron metabolism is disturbed during acute episodes of malaria 12 and the possible effects of recurrent, asymptomatic malaria infections on iron metabolism need further investigation. Studies with chromium-labelled RBC
have shown that splenic clearance of nonparasitized RBC is enhanced for some time after an episode of acute, symptomatic malarial,13 perhaps due to hypersplenism. Splenic enlargement is found frequently in healthy children in malarious areas and this might be associated with an enhanced rate of clearance of non-parasitised RBC. I am unaware of any study in which this has been specifically investigated, but there is little doubt that hypersplenism is an important cause of anaemia (and sometimes leucopenia and thrombocytopenia) in patients with the hyper-reactive malarial splenomegaly syndrome (see below). The idea that immunological factors play an important part in the pathogenesis of malarial anaemia was once fashionable but is now held less widely. Early studies showed that sera from subjects resident in malarious areas frequently contained heterophile antibodies, cold agglutinins, and antibodies that reacted with altered human RBC 14. However, there is no evidence that any of these antibodies are directly haemolytic in vivo. Several studies demonstrated positive direct anti-globulin tests (Coombs tests) in some patients with malaria. For example, a series of studies in The Gambia found positive Coombs tests in approximately 50% of healthy village children and in a similar proportion of patients with acute, clinical malaria 15-]7. But positive Coombs tests cor_
209
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related only weakly with the presence of parasitaemia or anaemia 15. Another study of Gambian patients with P. falciparum malaria found no correlation between anaemia and a positive antiglobulin test 18 and, in Thai patients with acute malaria, sensitisation of RBC could not be demonstrated 19. In Gambian children 15, positive antiglobulin tests were due most frequently to bound C3d. IgG was present less often 15. When present, bound IgG was usually of the IgG2 or IgG4 sub-classes but anaemia was related only to the ]presence of bound IgG117. Immunoglobulin eluted from IgGpositive RBC contained antibody to P. falciparum schizonts - compatible with the idea that erythrocyte sensitisation resulted from passive attachment to RBC of complement fining, malaria antigen-antibody complexes 16. Attempts have been made to quantify the amount of immunoglobulin present on the surface of RBC obtained from malaria patients with a positive anti-globulin test. Nigerian patients showed some correlation between the amount of bound IgG and the presence of anaemia 2° but this was not the case in The Gambia where low levels of IgG binding (90-897 molecules per red cell) were noted 21. Much higher levels of binding would probably be required to facilitate erythophagocytosis. Thus, it appears that in the majority of cases where a positive antiglobulin test is found in patients with malaria, or in children resident in malarious areas, this is of no clinical consequence. In malaria endemic areas, anaemia is found most frequently in younger children suggesting that acute malaria infections play an important part in its pathogenesis. This idea is supported by the iact that successful malaria control campaigns have had a greater effect on haemoglobin levels or packed cell volumes (PC,V) in young children than in older children or adults23, 24. However in Tanzania, malaria control did have a small but sign~icant effect on haemoglobin levels in adults 23, and in The Gambia, we have found considerably higher PCVs in children given malaria prophylaxis than in children given treatment for acute infections alone (B.M. Greenwood et al. unpublished). Thus it is probable that acute and sub-clinical infections both contribute to the anaemia found frequently in children in areas where malaria is endemic. Malaria and the Outcome of Pregnancy Immunity to malaria -- acquired gradually throughout childhoc~i - may be lost during pregnancy, especially in primag-
Table 2. Evidence implicating malaria in the pathogenesis of the hyper-reactive malaria splenomegaly syndrome (supporting refs cited in Ref. 14) •
The condition is found only in areas where malaria is endemic.
•
Possessionof the haemoglobin genotype AS provides protection against the syndrome.
•
Patients with HPIS have higher levels of malaria antibodies than controls.
•
Hypergammaglobulinaemia and splenomegaly regress in patients who take malaria chemoprophylaxis.
ravidae. In semi-immune women, malaria infections during pregnancy rarely cause an acute febrile illness of the type seen in children, but can contribute to the anaemia of pregnancy and, occasionally, cause a severe, fife-threatening haemolytic anaemia25. Low-level, asymptomatic infections in the mother can cause parasitisation of the placenta26. This is important because it leads to lowering of the birth weight which has an adverse effect on child survival.
Splenomegaly Acute, clinical episodes of malaria can cause splenomegaly which regresses after the infection has been treated or resolved. But when malaria infections are recurrent, splenomegaly does not regress between attacks and a high proportion of children resident in malaria-endemic areas have modestly enlarged spleens. Whether these children are disadvantaged in any way is In a few subjects, binding of uncertain. As discussed above, it is possible large amounts oflgG I to that splenomegaly causes enhanced RBC especiallyif combined destruction of RBC but whether this is clin- with C3, may lead to enhanced RBC destruction. ically important is unknown. Spleens However, a recent report enlarged as a result of infection with P. that human RBC coated vivax seem more prone to rupture than nor- with IgG immune complexes mal spleens but this does not seem to be the are not taken up in vitro by phagocytic cells22 makes it case for P. falciparum. uncertain how this is brought In malaria endemic areas the prevalence about. of splenomegaly declines as immunity to malaria is acquired and, in most communities, few adults have enlarged spleens. However, in a proportion of subjects repeated exposure to malaria results in massive enlargement of the spleen and overproduction of IgM and malaria antibodies. This condition - originally termed the tropical splenomegaly syndrome (TSS) - is now called the hyper-reactive malarial splenomegaly syndrome (HMS) 27 to differentiate it from other forms of massive splenomegaly of unknown cause28,29. HMS is found only in areas where malaria is endemic but the prevalence of the syndrome varies considerably from region to region. In some villages in Papua New Guinea and in Indonesia a high proportion of the adult population are affected30, yet the condition is generally rare
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Genetic factors are probably important in the development of riMS. In West Africa the syndrome is found more frequently amongst Fulas (Fulanis) than other ethnic groups2&31, In Papua New Guinea4s there is some association between HMS and presence of the HLA antigen DR2.
Fig. 3. A schematic representation of the possible pathogenesis of the hyper-reactive malarial splenomegaly syndrome.
in areas of West Africa with a similar malaria endemicity31. The predominant clinical feature of HMS is massive enlargement of the spleen which may cause local discomfort32. Some degree of anaemia is usually found which is due partly to hypersplenism and partly to hypervolaemia. Acute haemolytic crises may occur. Liver biopsy usually shows infiltration of hepatic sinusoids with lymphocytes but this is not an invariable finding nor is it specific for HMS. For reasons that are not understood, patients with HMS have a poor prognosis. Nearly 50% of a large group of patients studied in Papua New Guinea died during a 15 year follow up period 33, and a similar high mortality has been noted in Nigeria and The Gambia (Ref. 34 and B.M. Greenwood et al. unpublished). Patients with HMS have very high serum levels of IgM and immune complexes which possibly represent immunoglobulin aggregates rather than true antigen/antibody complexes35. Clearance of these complexes is probably the main reason for the massive splenomegaly36. Cell mediated immune responses are usually normal; infiltration of
hepatic sinusoids with T-lymphocytes may represent a cellular immune response to antigens present in Kuppfer cells37. The pathogenesis of HMS is still only partially understood (Fig. 3) but there is strong evidence (Table 2) that malaria has a dominant role. Malaria is a powerful stimulus to immunoglobulin production and, amongst normal subjects resident in malarious areas, serum levels of IgM and autoantibodies rise throughout life. It seems likely that suppressor mechanisms are normally activated to overcome this malariadriven immune hyper-reactivity, but these are probably defective in patients with HMS. Studies in Indonesia38 have shown that patients with HMS have reduced numbers of T8 (suppressor) cells and normal levels of T4 (helper) cells, giving a high T4/ T8 ratio. In vitro studies show that IgM production by B-cells obtained from patients with HMS can be suppressed by normal T8 cells38 - strongly suggesting that the abnormality in patients with HMS lies in their T8 lymphocyte sub-population. Sera of Indonesian patients with HMS contain an antibody that can lyse T8 cells in the presence of complement; this autoantibody39, perhaps induced by malaria, may be the cause of the poor T-cell suppressor function found in patients with HMS. Lack of control over B-cell proliferation is probably the basic immunological defect in HMS, but it is not certain that this is brought about in all patients in the same way. The prevalence of HMS differs between Indonesia and Papua New Guinea, and West Africa, and there are differences in the clinical features of the syndrome in these areas. West African patients with HMS sometimes have a lymphocytosis which may be marked enough to suggest a diagnosis of leukaemia4°, whereas this is rarely, if ever, encountered in Indonesian or Papuan patients. It is important that the elegant in vitro studies on immunoglobulin production undertaken in Indonesian patients with HMS should be repeated in Africa. There is clear evidence that repeated subclinical infections of malaria are more important than acute symptomatic infections in causing HMS. Thus, HMS is primarily a disease of adults rather than of children, and the clinical and immunological features of the syndrome recur rapidly in adults brought into remission with chemoprophylaxis who stop taking their treatment.
Nephrotic Syndrome The incidence of the nephrotic syndrome
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in children is much higher in some parts of the tropics than in countries with a temperate climate. In part, tiffs is because the syndrome can result from infection with P. malariae (Table 3). The malarial nephrotic syndrome is seen most frequently in children aged 5-10 years, but it also occurs in adults - especially in East Africa42. Renal biopsies from children with the malarial nephrotic syndrome in Nigeria, showed thickening of the capillary basement membrane with focal and segmental sclerosis of glomerular tufts. On electron microscopy, sub-endothelial deposits and lacunae within the basement membrane were seen43,44. The prognosis of children with the malarial nephrotic syndrome is poor and most progress to chronic renal, failure and death, despite treatment with corticosteroids or immunosuppressive agents. The P. malariae nephrotic syndrome is, at least in part, an immune-complex nephritis. Studies in Nigeria and Uganda have shown deposits of in~nunoglobulin and complement in the glomerular basement membrane of nearly all cases4~,46. The pattern of immunoglobulha staining seen on immunofluorescence varies from a nodular to a more linear pattern, and in about one third of cases, P. mala~iae antigen has been demonstrated within basement membrane deposits. Yet despite these characteristic immunological changes,the pathogenesis of quartan malaria nephropathy is not straightforward. Unlike most other forms of infection-related immune-complex nephritis, the condition does not resolve when malaria is treated and reinfection prevented by chemoprophylaxis. This suggests that renal damage may be sustained by autoimmune mechanisms but this possibility has not been investigated adequately. What proportion ofP. malariae infections leads to renal damage, and why this happens in some children but not in others, remain unknown. In semi-immunes, P'. malariae infections rarely cause acute clinical illness with high levels of parasitaemia. Most lead to asymptomatic infections with persistent, low-level parasitaemia. Thus it is likely that these asymptomatic infections, rather than acute clinical episodes, are the main cause of the P. malariae nephrotic syndrome. This concept is supported by data from the malaria eradication campaign in Guyana47. Before eradication, chronic nephritis accounted for 7.8% of deaths in children and 13.5% of deaths in adults, among whom acute clinical episodes of malaria would have been infrequent. Following
21 I
Table 3. Evidence implicating P. malariae in the pathogenesis of the nephrotic syndrome (supporting refs cited in Ref. 14) •
The age distribution of the nephrotic syndrome in West Africa parallels that of P. malariae infection.
P. malariae is found 5 times more frequently in African patients with the nephrotic syndrome, than in controls. In Africa, patients with the nephrotic syndrome have higher levels of antibody to P. malariae, than controls.
P. malariae antigens can be demonstrated in glomerular deposits in about one third of African patients with the nephrotic syndrome. The nephrotic syndrome can be induced experimentally by infecting monkeys with P. malariae. Eradication of malaria in Guyana led to a fall in the incidence of the nephrotic syndrome.
eradication, chronic nephritis ceased to be a cause of death in children and accounted for only 2.5% of deaths in adults.
Burkitt's Lymphoma Burkitt's lymphoma is one of the commonest causes of childhood cancer in tropical Africa and some other parts of the tropics. In these areas it occurs in approximately 1 per 10 000 children per year4s. The tumour is seen most frequently in 2-16 year old children, with a mean age at presentation of about 7 years. A tumour of the jaw is the most usual presenting feature but multiple tumours are common and may occur in the abdomen and at other sites. Histological examination shows a characteristic 'starry sky' appearance of macrophages against a background of undifferentiated, round lymphocytes. The malignant lymphocytes of Burkitt's lymphoma have B-cell markers 49 and the tumour is a monoclonal B-cell lymphoma. Spontaneous regression occasionally occurs and, except in the most advanced stages, the tumour responds well to chemotherapy. Cytogenetic studies have shown that all, or nearly all, Burkitt's tumours show chromosomal translocationsS0, 51. The corn-
Some authors believe that the histological changes associated with P. malanae nephrotic syndrome are su~ciently characteristic to justify the designation of 'quartan malaria nephropathy', In Uganda however, more variable histological changes have been reported.
Table 4. Evidence implicating infection with Epstein-Barr virus (EBV) in the pathogenesis of Burkitt's lymphoma EBV DNA is integrated into the nuclear DNA of nearly all African cases of Burkitt's lymphoma. •
Children who subsequently develop Burkitt's lymphoma have higher levels of antibody to EBV viral capsid antigen (VCA) than controls.
•
EBV can 'immortalize' B-cells in in vitro cultures.
•
EBV can induce lymphomas in tamarind monkeys.
•
TamarindscanbeprotectedagainsttheoncogeniceffectsofEBVinfectionby vaccination.
212
Parasitology Today, vot. 3, no. 7, 1987
Table 5. Evidence implicating malaria in the pathogenesis of Burkitt's lymphoma (supporting refs cited in Ref. 14) •
A high incidence of Burkitt's lymphoma is found only in areas where malaria is endemic.
•
Within malarious areasthe incidence of Burkitt's lymphoma is low in areaswhere malaria infection is naturally infrequent or has been controlled.
and differentiation52. Fusion of c-myc with an Ig heavy chain constant gene appears to cause loss of normal growth regulation and uncontrolled proliferation of B-lymphocytes.
The Epstein Barr virus (EBV) plays an important part in the aetiology of Burkitt's lymphoma (Table 4). However, EBV is not • The haemoglobin genotype AS is under-represented in patients with Burkitt's essential to the pathogenesis of Burkitt's lymphoma. tumour and histologically indistinguishable • Malaria infection of experimental animals can enhance the oncogenic potential of lymphomas have been described in subjects tumour viruses. with no evidence of EBV infection. Infection with EBV is universal but a high incimonest abnormalities are translocations dence of Burkitt's lymphoma is found only between part of the long arm of chromo- in tropical areas with a high prevalence of some 8 and chromosomes 2, 14 or 22 which malaria - suggesting that malaria plays are all loci ofimmunoglobulin genes. These some part in its pathogenesis (Table 5). rearrangements are almost exclusive to BThere are two main hypotheses for the cell lymphomas. The translocated part of role of malaria in the pathogenesis of Burchromosome 8 contains an oncogene (c-myc) kitt's lymphoma. One postulates that by which normally influences cellular growth stimulating immunoglobulin-producing cells, malaria increases the likelihood of a chromosomal translocation at the site of one of the genes controlling immunoglobulin production, thus causing the transfer of the c-myc oncogene to an area where it permits unrestrained growth of B-cells53. P. falciparum can cause immortalization of Blymphocytes in culture but it is not known whether these transformed cells show chromosomal translocations 54. The primary event could be activation of B-cells by malaria leading to chromosomal translocation, followed by EBV infection and consequent immortalization of the transformed cells52, or it could be primary EBV infection and immortalization of B-ceUs subsequently stimulated by repeated malaria infections and thus increasing the likelihood of chromosomal translocation53 (Fig. 4). A second hypothesis suggests that malaria impairs the host's ability to control the growth of EBV-infected immortalized B-cells once these have been formed. In The Gambia it was shown that the ability of Tlymphocytes to control the growth of EBVinfected B-cells was impaired in children with acute malaria, but control was regained on recovery55. This loss of suppressor activity was associated with a change in the T4/T8 lymphocyte ratio. In Papua New Guinea, lymphocytes from subFig. 4. Two possible mechanisms for the interaction of malaria and Epstein Barr Virus (EBV) in the pathogenesis of Burkitt' s lymphoma. The lymphoma seems to be caused by jects resident in malarious areas were less uncontrolled growth of B-cells infected with EBV, which show a chromosomal transloca- effective at controlling the growth of EBVtion affecting cellular growth and differentiation. The inducer-suppressor T-cell chain is infected B-cells in vitro, compared to lyminhibited by malaria, allowing the tumour to develop. phocytes from residents of non-malarious Malaria also acts in the initiation of the B-cell transformation. In hypothesis A, the areas 56. B-cells are first immortalized by infection with EBV, and their multiplication is then The relative contributions of acute and stimulated by repeated malaria infections - thus increasing the likelihood of sub-clinical malaria infections to the chromosomal translocation. In hypothesis B, the primary event would be malaria infection promoting B-cell multiplication and chromosomal translocation, followed by pathogenesis of Burkitt's lymphoma is difEBV infection which immortalizes the transformed cells. ficult to assess. The age distribution of the
Parasitology Today, vol. 3, no. 7, 1987
tumour - maximum in children in the age range at which severe clinical infections are frequent - suggests that: these may be the most important factor but interpretation is complicated by the role of EBV infection which is also age-related. And if loss of Tcell control of EBV-infected B-cells is an important pathogenic factor, then sub-clinical infections could be important. The studies in Papua New Guinea which showed impaired T-cell control over EBVinfected B-cells were canied out in adults in whom acute episodes of malaria would have been unlikely. Immunomodulation Malaria is both immunostimulatory and immunosuppressive57. In endemic areas, healthy individuals have high serum IgG and IgM, and raised titres of rheumatoid factor and other autoantibodies 14. How malaria causes polyclonal B-cell activation is not fully understood, but repeated exposure to parasites with variant antigens and the production of mitogens by the parasites, are probably both involved. Yet despite immunological over-reactivity, some immune functions are suppressed in patients with malaria. Antibody responses to a variety of antigens are impaired - particularly to poorly immunogenic antigens such as capsular polysaccharides58. Do these immunological changes have any clinical importance? There is evidence in experimental animals that malaria increases susceptibility to some other infections and we have shoran recently that P. falciparum malaria predisposes to Salmonella infections in Gambian children 59. It is also possible that the immunomodulation produced by malaria could influence susceptibility to immune-mediated noninfectious diseases but this is difficult to investigate. Some evidence to support the idea that malaria has art indirect effect on mortality from other conditions is given by intervention studies. In 3 successful malaria control schemes in Africa, infant and child mortality were reduced by a greater extent than predicted from previous death rates attributed to malaria 6°. At Pare-Taveta in Tanzania and Kisumu in Kenya - both areas of high malaria endemicity - successful control of malaria with insecticides led to a reduction of mortality in adults as well as in children, suggesting that in these communities sub-clinical :malaria infections contributed in some indirect way to adult mortalitytl,62. This idea is also supported by results from Guyana where mortality from pneumonia, as well as from nephritis, fell in
213
adults as well as in children as malaria came under control47.
Long-term Consequences of Malaria Consideration of the long-term consequences of malaria and the part played by subclinical infections allows us to make some guesses about what might happen in a community protected from acute, clinical malaria by a partially effective vaccine. My guess is that the prevalence of Burkitt's lymphoma and - if a partially protective P. malariae vaccine was introduced - of the malaria nephrotic syndrome, would decline but that neither condition would disappear. Some improvement in the overall haemoglobin level of the population might be anticipated. I would be surprised if a partially protective vaccine had much effect on the incidence of the hyper-reactive malaria splenomegaly syndrome, but in most malaria endemic areas, Burkitt's lymphoma, quartan malaria nephropathy and HMS are relatively infrequent complications of malaria. Persistence of these conditions might be a low price to pay for the booster immunizing effect of asymptomatic malaria infections in subjects given a malaria vaccine that was only partially protective. References
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Antibacterial Action of Myiasiscausing Flies G.R, Erdmann Some species of calliphorid blowflies* lay their eggs in wounds; their larvae develop by feeding on the tissue, and the infection is known as myiasis or fly-strike. But wounds, from whatever cause, are frequently contaminated with bacteria - many of which can spread in the bloodstream causing septicaemia and~or toxaemia. For example, wound contamination with Clostridium welchii - leading to 'gas gangrene' - was a frequent cause of death amongst battlefield casualties. It is from such situations that early observations were made on the beneficial effect of sorne blow~y larvae in limiting the bacterial infection of wounds. Indeed, some military surgeons would deliberately infest wounds with blowfly maggots in order to prevent bacterial complications. Now, a century or two later, the search for new antibiotics had led researchers back to these early observations, and in this article, Gary Erdmann des- ~ ...... tribes progress in understanding the antibacterial action of blowfly maggots.
Battlefield casualties frequently died from septicaemia following bacterial infection of wounds. Surgeonsin Napoleon's Armies noted, however, that wounds infested with blowfly maggots often did not go septic ~ resulting in higher survivorship of wounded soldiers. ~ ~ ?
iii~ii
¸¸
A
One of the first observations of wound infestations was by D.J. Larrey, a military surgeon in Napoleon's army I. He recorded that larvae of a 'blue fly' common in S' (possibly Chrysomyia or Wohlfahrtia; see Refs 2-4) frequently invaded the wounds of soldiers while on thehe Alfi battlefield. Presence of the larvae helped / to remove scar tissue and reduced the threat of bacterial m infection. During the US civil war. J.F.
[]
*Formerly, the family Calliphoridae included the subfamilies Calliphorinaeand Sacrophaginae; species of both groups lay their eggs in damaged, decayingor dead verLebrate1issues.More recently, both subfamilieshavebeenelevatedto familyrank. ~)1987, Elsevier Publications,Cambridge 0169-4758/87/$0200