C/in. exp. Immunol. (1979) 35, 119-127.
Direct Coombs antiglobulin reactions in Gambian children with Plasmodium fakiparum malaria I. INCIDENCE AND CLASS SPECIFICITY CHR I S T I NE A. FACER, R. S. B R AY * & J. BROWN * Department of Haematology, The London Hospital Medical College, London and *Medical Research Council Laboratories, Fajara, The Gambia, West Africa
(Received 26 July 1978) SUMMARY
Gambian children with past or present Plasmodium falciparum malaria were investigated for the incidence of Coombs positivity using monospecific antisera. Approximately 5000 were positive and the most frequent form of erythrocyte sensitization was with C3d. Other specificities, EIgG, EIgGC3d and EIgGC4bC3bC3d were less common. Erthyrocytes were never found sensitized with IgA or IgM. There was no correlation between a positive test and age, tribal status or level of parasitaemia at presentation, although a positive test was often found in association with anaemia. Sensitized erythrocytes were present in the circulation for a period of up to 6 weeks following initial observation. The mechanism of erythrocyte sensitization is not known, but the results suggest a Type III complex-mediated hypersensitivity involving parasite antigen-antibody complexes. It is likely that these reactions contribute to the pathogenesis of the anaemia in falciparum malaria. INTRODUCTION The degree of anaemia in malaria infections is frequently disproportional to the degree of parasitaemia, and it has been questioned as to whether additional immunological mechanisms involving non-parasitized erythrocytes might account for this (Zuckerman, 1964; Brown, 1969). Recent attention has focused on Type II and/or Type III cytotoxicity in the search for additional explanations for the anaemia (Coombs, 1976). The finding of positive Coombs antiglobulin tests in a few patients with malaria strengthened these possibilities (Zoutendyke & Gear, 1951; Demirag & Sozer, 1956; Barrett-Connor, 1967; Topley, Knight & Woodruff, 1973). However, there are reservations about these reports because of the nonspecificity of the antiglobulin reagents used which, since they included anti-transferrins, gave falsepositive tests when reticulocytosis was evident (Coombs, 1976). Likewise, the negative Coombs tests reported in malaria (Rosenberg et al., 1973) may be a result of inadequately diluted reagents, resulting in prozone phenomenon during the haemagglutination test (Pirofsky, 1969). In this report we describe results of direct Coombs antiglobulin tests on a large group of Gambian children with past or present infections with P. falciparum using monospecific antisera to human immunoglobulins and complement components. The results indicate that sensitization of non-parasitized erythrocytes in some children contributes to the pathogenesis of anaemia in this infection. MATERIALS AND METHODS Patients. Three groups of children were investigated during the 1976 rainy season of The Gambia (August-November): (1) eighty-five children (aged between 2 months and 10 years) attending the Out-Patient's Clinic at the Medical Research Council Laboratories at Fajara with clinical and parasitological features of acute malaria. All the children had Plasmodium falciparum infections with parasitaemias ranging from 1% to 40% parasitized erythrocytes (E). Many were anaemic with a Correspondence: Dr Christine A. Facer, Department of Haematology, The London Hospital Medical College, Turner Street, London El 2AD. 0099-9104/79/0010-0119$02.00 (C 1979 Blackwell Scientific Publications
119
120
Christine A. Facer, R. S. Bray & J. Brown
packed cell volume (PCV) of less than 30%o, but none required transfusion. Medical histories were obtained and the complete physical examination of anaemic children suggested that malaria was the sole cause of the anaemia. Malaria patients with concommitant malnutrition, or viral, bacterial or other parasitic disease, or who had been given anti-malarials were excluded from the study. Immediately after the initial blood sample had been collected, all children with a parasitaemia greater than 2% were treated with oral chloroquine (50 mg base). A second or third blood sample was obtained from twenty-one of these children in whom follow-up was achieved 1-6 weeks after presentation. (2) Seventy-five children of the same age group as group (1) were selected at random from the rural village population of Brefet. This group served as a control for the acute disease since, although parasitaemias were common during the 6 week study period, they were always minimal and only on one occasion exceeded a 100 parasitaemia. Gametocyte carriers predominated. (3) Two children (MH and JM) were admitted to the MRC ward because of severe anaemia and convulsions. A blood sample was obtained before chloroquine treatment and follow-up samples were taken at intervals during the week following admission. All blood samples were collected into ethylenediamine tetracetic acid (EDTA; 20 mM) and maintained between 20 and 30'C before performing the antiglobulin test within 2 hr ofcollection. Haematology. PCV, haemoglobin (Hb), differential white cell counts and reticulocytes were determined using standard procedures (Dacie & Lewis, 1975). Parasitaemia. Thick and thin blood films were stained with Giemsa and parasitaemia was quantified by counting the number of parasitized E per 500 E expressed as a percentage. Direct antiglobulin test (DAT). Commercially available antisera (Hoechst Pharmaceuticals) to human immunoglobulins (Igs) with ca, y and j chain specificity were used in addition to antisera to human C3b/C3c (131C/131A) and C4b (P1IE). AntiC3d (a2D) serum was a gift from Professor P. L. Mollison. Antisera were heat-inactivated at 56'C for 30 min and absorbed with equal volumes of packed washed blood group 0 and AB erythrocytes with Rh R1R2 phenotype at 37°C and 4°C. The specificity of the antisera was checked by a haemagglutination test using E sensitized in vitro with purified IgA, IgG, IgM and complement (C') components (Freedman & Mollison, 1976). The DAT was performed in microtitre 'V' plates using doubling dilutions of monospecific antisera in isotonic saline starting at a I dilution. We found the use of this micromethod increased the sensitivity of the reaction, avoided false negative results due to prozone particularly with the anti-IgG reagent, and from the titre gave an indication of the relative strength of E sensitization. Patients' E were washed four times in saline at room temperature and 0 25 pl of a 2.5% suspension added to each well of the plates. Plates were shaken, incubated for 1 hr at 37°C and agglutination scored from 4+ to negative. The titre recorded was the highest dilution giving a 2+ reaction. Controls included normal unsensitized adult Gambian group 0 R1R2 E, and the same cells sensitized with anti-D (Dacie & Lewis, 1975). Statistical evaluation. Statistical evaluation was carried out using the Z2 test.
RESULTS Incidence and class specificity A high proportion of the 160 children investigated had a positive DAT. Fig. 1 shows comparative results from the Fajara and Brefet children. In the latter group, fifty-five of the seventy-five children examined had a P. falciparum parasitaemia and 56% of these were DAT positive. The remaining twenty children with negative parasitaemias at each weekly investigation over a period of 6 weeks always had E negative in the DAT. The use of monospecific antisera detected five main groups of E sensitization (Fig. 1), the most frequent being E sensitized with conglutinogen activating factor (KAF) reacted C3b, C3d. E were never found sensitized with IgA or IgM. In the four cases with EIgGC4bC3bC3d sensitization, patients were severely anaemic with a PCV range of 15-24% (Hb 5-7°O). E taken from six healthy Gambian adults were negative in the DAT. Association with age, sex and tribe Children of all ages and tribes were equally affected. A higher proportion of male as opposed to female children in both the Fajara and Brefet village group had a positive DAT (12% and 14% respectively). The significance of this finding is unknown.
Association with parasitaemia To examine the possibility that the level of parasitaemia at presentation might be related to the DAT result, children with acute P. falciparum malaria (from Fajara) were divided into five main groups according to the percentage parasitaemia. Fig. 2 shows that most children examined had a parasitaemia of 1-6% parasitized E and approximately 50%/O had a positive DAT. Very high parasitaemias were not
Antiglobulin reactions in malaria
121
a-
0
Q) 0 CIL
A
B
C
D
E
T
Specificity
FIG. 1. Class specificity of the DAT in children with acute falciparum malaria (Fajara) (a) and low grade chronic infections (Brefet) (E). A = EIgG, B = EC3d, C = EIgGC3d, D = EC4bC3bC3d, E = EIgGC4bC3b C3d, and T = total positive (all classes).
necessarily coincident with a positive test. A slightly lower percentage of positives (30%) was found among patients with < 1% parasitaemia. This was in contrast to the Brefet village study where all children had a < 1% parasitaemia, but 56% were Coombs positive. All positive DAT reactions found in patients from Fajara with a parasitaemia < 1% were specific for C3d.
Association with anaemia Children from Fajara and Brefet were divided into two groups, those anaemic with a PCV of < 30% 40
30-
20 E
z
Io
0 I Fajara; P> 04 Brefet). Forty-four per cent of the Fajara children with acute malaria were anaemic and Table 1 shows that there was a higher percentage of DAT positive reactions in the anaemic group and this was statistically highly significant (X2 = 15-7; P< 0001). However, the difference was not statistically significant in the Brefet group (X2 = 1-15; P> 0 1) and in both groups there were a large number of patients who had a positive test, but no anaemia. That these observations related to the specificity of E sensitization is shown in Table 2. Two significant observations were made. All four patients with active C3 components on the E were severely anaemic with reticulocytosis and circulating normoblasts, and C4b was always demonstrated along with C3b sensitization. This correlated with the finding of monocyte erythrophagocytosis of non-parasitized E in peripheral blood films of these patients (Fig. 3). Erythrophagocytosis by polymorphonuclear cells was never observed although malarial pigment was often seen in neutrophils and basophils. There was no association between the antiglobulin titre and anaemia (Table 3).
Close follow-up study Haematological and parasitological findings of the two children in this study are shown in Fig. 4. In patient JM, IgG appeared on the erythrocytes the day following chloroquine chemotherapy, followed by a decrease in PCV and Hb. However, both PCV and Hb returned to normal during the following week, although EIgG could still be demonstrated. The high initial parasitaemia of this patient might therefore explain the drop in PCV after chloroquine chemotherapy. Patient MH, with initial low paraTABLE 2. Specificity of DAT as related to anaemia in acute P. fakiparum malaria
Specificity of E sensitization (%o) IgG Fajara children Anaemic
C3d
+ C3d
0
69-2 (19)
15-3
15.3
(4)
(4)
8.3
75-0
16-6
0
(1)
(9)
(2)
(0)
(0) Not anaemic
IgG + C3d C3b
+ C4b +
IgG
Figures in parentheses are the number ofindividuals in each group. The child with EC4bC3bC3d was included in the last group.
123
Antiglobulin reactions in malaria
FIG. 3. Erythrophagocytosis by monocytes in the peripheral blood of a child with falciparum malaria and (Magnification x 2000.)
severe anaemia.
sitaemia, was anaemic when admitted but became severely anaemic following chloroquine chemotherapy, and the sharp drop in PCV and Hb coincided with the appearance of E-bound active C3 and C4. Likewise the disappearance of active C3 and C4 led to a marked recovery in PCV and Hb. Long-term fllow-up study Alterations in DAT specificity following chemotherapy (Fajara) and observations over a 6 week study period (Brefet) are shown in Tables 4 and 5. A number of differences were observed between the two groups. The majority of the patients with acute malaria who had a positive DAT on presentation, remained positive following chemotherapy, most retaining the same specificity (Table 4). A much lower percentage in the Brefet group followed this pattern (Table 5). A greater percentage of the Brefet children who had a negative DAT on presentation remained negative when compared to the Fajara group.
TABLE 3. DAT titres from children with acute P. fakiparum malaria with or without anaemia DAT log2 titre
Fajara children Anaemic Not anaemic
IgG
C3d
C3b
C4b
7-3±0-6 7-2±0-2
4-0±0-2
1-2±0-2 0
1-3±0-2
4 0±0 5
Log2 titres given as a meanl 1 s.e.
0
124
Christine A. Facer, R. 30
a-
a-
Direct Coombs antiglobulin reactions in Gambian children with Plasmodium fakiparum malaria I. INCIDENCE AND CLASS SPECIFICITY CHR I S T I NE A. FACER, R. S. B R AY * & J. BROWN * Department of Haematology, The London Hospital Medical College, London and *Medical Research Council Laboratories, Fajara, The Gambia, West Africa
(Received 26 July 1978) SUMMARY
Gambian children with past or present Plasmodium falciparum malaria were investigated for the incidence of Coombs positivity using monospecific antisera. Approximately 5000 were positive and the most frequent form of erythrocyte sensitization was with C3d. Other specificities, EIgG, EIgGC3d and EIgGC4bC3bC3d were less common. Erthyrocytes were never found sensitized with IgA or IgM. There was no correlation between a positive test and age, tribal status or level of parasitaemia at presentation, although a positive test was often found in association with anaemia. Sensitized erythrocytes were present in the circulation for a period of up to 6 weeks following initial observation. The mechanism of erythrocyte sensitization is not known, but the results suggest a Type III complex-mediated hypersensitivity involving parasite antigen-antibody complexes. It is likely that these reactions contribute to the pathogenesis of the anaemia in falciparum malaria. INTRODUCTION The degree of anaemia in malaria infections is frequently disproportional to the degree of parasitaemia, and it has been questioned as to whether additional immunological mechanisms involving non-parasitized erythrocytes might account for this (Zuckerman, 1964; Brown, 1969). Recent attention has focused on Type II and/or Type III cytotoxicity in the search for additional explanations for the anaemia (Coombs, 1976). The finding of positive Coombs antiglobulin tests in a few patients with malaria strengthened these possibilities (Zoutendyke & Gear, 1951; Demirag & Sozer, 1956; Barrett-Connor, 1967; Topley, Knight & Woodruff, 1973). However, there are reservations about these reports because of the nonspecificity of the antiglobulin reagents used which, since they included anti-transferrins, gave falsepositive tests when reticulocytosis was evident (Coombs, 1976). Likewise, the negative Coombs tests reported in malaria (Rosenberg et al., 1973) may be a result of inadequately diluted reagents, resulting in prozone phenomenon during the haemagglutination test (Pirofsky, 1969). In this report we describe results of direct Coombs antiglobulin tests on a large group of Gambian children with past or present infections with P. falciparum using monospecific antisera to human immunoglobulins and complement components. The results indicate that sensitization of non-parasitized erythrocytes in some children contributes to the pathogenesis of anaemia in this infection. MATERIALS AND METHODS Patients. Three groups of children were investigated during the 1976 rainy season of The Gambia (August-November): (1) eighty-five children (aged between 2 months and 10 years) attending the Out-Patient's Clinic at the Medical Research Council Laboratories at Fajara with clinical and parasitological features of acute malaria. All the children had Plasmodium falciparum infections with parasitaemias ranging from 1% to 40% parasitized erythrocytes (E). Many were anaemic with a Correspondence: Dr Christine A. Facer, Department of Haematology, The London Hospital Medical College, Turner Street, London El 2AD. 0099-9104/79/0010-0119$02.00 (C 1979 Blackwell Scientific Publications
119
120
Christine A. Facer, R. S. Bray & J. Brown
packed cell volume (PCV) of less than 30%o, but none required transfusion. Medical histories were obtained and the complete physical examination of anaemic children suggested that malaria was the sole cause of the anaemia. Malaria patients with concommitant malnutrition, or viral, bacterial or other parasitic disease, or who had been given anti-malarials were excluded from the study. Immediately after the initial blood sample had been collected, all children with a parasitaemia greater than 2% were treated with oral chloroquine (50 mg base). A second or third blood sample was obtained from twenty-one of these children in whom follow-up was achieved 1-6 weeks after presentation. (2) Seventy-five children of the same age group as group (1) were selected at random from the rural village population of Brefet. This group served as a control for the acute disease since, although parasitaemias were common during the 6 week study period, they were always minimal and only on one occasion exceeded a 100 parasitaemia. Gametocyte carriers predominated. (3) Two children (MH and JM) were admitted to the MRC ward because of severe anaemia and convulsions. A blood sample was obtained before chloroquine treatment and follow-up samples were taken at intervals during the week following admission. All blood samples were collected into ethylenediamine tetracetic acid (EDTA; 20 mM) and maintained between 20 and 30'C before performing the antiglobulin test within 2 hr ofcollection. Haematology. PCV, haemoglobin (Hb), differential white cell counts and reticulocytes were determined using standard procedures (Dacie & Lewis, 1975). Parasitaemia. Thick and thin blood films were stained with Giemsa and parasitaemia was quantified by counting the number of parasitized E per 500 E expressed as a percentage. Direct antiglobulin test (DAT). Commercially available antisera (Hoechst Pharmaceuticals) to human immunoglobulins (Igs) with ca, y and j chain specificity were used in addition to antisera to human C3b/C3c (131C/131A) and C4b (P1IE). AntiC3d (a2D) serum was a gift from Professor P. L. Mollison. Antisera were heat-inactivated at 56'C for 30 min and absorbed with equal volumes of packed washed blood group 0 and AB erythrocytes with Rh R1R2 phenotype at 37°C and 4°C. The specificity of the antisera was checked by a haemagglutination test using E sensitized in vitro with purified IgA, IgG, IgM and complement (C') components (Freedman & Mollison, 1976). The DAT was performed in microtitre 'V' plates using doubling dilutions of monospecific antisera in isotonic saline starting at a I dilution. We found the use of this micromethod increased the sensitivity of the reaction, avoided false negative results due to prozone particularly with the anti-IgG reagent, and from the titre gave an indication of the relative strength of E sensitization. Patients' E were washed four times in saline at room temperature and 0 25 pl of a 2.5% suspension added to each well of the plates. Plates were shaken, incubated for 1 hr at 37°C and agglutination scored from 4+ to negative. The titre recorded was the highest dilution giving a 2+ reaction. Controls included normal unsensitized adult Gambian group 0 R1R2 E, and the same cells sensitized with anti-D (Dacie & Lewis, 1975). Statistical evaluation. Statistical evaluation was carried out using the Z2 test.
RESULTS Incidence and class specificity A high proportion of the 160 children investigated had a positive DAT. Fig. 1 shows comparative results from the Fajara and Brefet children. In the latter group, fifty-five of the seventy-five children examined had a P. falciparum parasitaemia and 56% of these were DAT positive. The remaining twenty children with negative parasitaemias at each weekly investigation over a period of 6 weeks always had E negative in the DAT. The use of monospecific antisera detected five main groups of E sensitization (Fig. 1), the most frequent being E sensitized with conglutinogen activating factor (KAF) reacted C3b, C3d. E were never found sensitized with IgA or IgM. In the four cases with EIgGC4bC3bC3d sensitization, patients were severely anaemic with a PCV range of 15-24% (Hb 5-7°O). E taken from six healthy Gambian adults were negative in the DAT. Association with age, sex and tribe Children of all ages and tribes were equally affected. A higher proportion of male as opposed to female children in both the Fajara and Brefet village group had a positive DAT (12% and 14% respectively). The significance of this finding is unknown.
Association with parasitaemia To examine the possibility that the level of parasitaemia at presentation might be related to the DAT result, children with acute P. falciparum malaria (from Fajara) were divided into five main groups according to the percentage parasitaemia. Fig. 2 shows that most children examined had a parasitaemia of 1-6% parasitized E and approximately 50%/O had a positive DAT. Very high parasitaemias were not
Antiglobulin reactions in malaria
121
a-
0
Q) 0 CIL
A
B
C
D
E
T
Specificity
FIG. 1. Class specificity of the DAT in children with acute falciparum malaria (Fajara) (a) and low grade chronic infections (Brefet) (E). A = EIgG, B = EC3d, C = EIgGC3d, D = EC4bC3bC3d, E = EIgGC4bC3b C3d, and T = total positive (all classes).
necessarily coincident with a positive test. A slightly lower percentage of positives (30%) was found among patients with < 1% parasitaemia. This was in contrast to the Brefet village study where all children had a < 1% parasitaemia, but 56% were Coombs positive. All positive DAT reactions found in patients from Fajara with a parasitaemia < 1% were specific for C3d.
Association with anaemia Children from Fajara and Brefet were divided into two groups, those anaemic with a PCV of < 30% 40
30-
20 E
z
Io
0 I Fajara; P> 04 Brefet). Forty-four per cent of the Fajara children with acute malaria were anaemic and Table 1 shows that there was a higher percentage of DAT positive reactions in the anaemic group and this was statistically highly significant (X2 = 15-7; P< 0001). However, the difference was not statistically significant in the Brefet group (X2 = 1-15; P> 0 1) and in both groups there were a large number of patients who had a positive test, but no anaemia. That these observations related to the specificity of E sensitization is shown in Table 2. Two significant observations were made. All four patients with active C3 components on the E were severely anaemic with reticulocytosis and circulating normoblasts, and C4b was always demonstrated along with C3b sensitization. This correlated with the finding of monocyte erythrophagocytosis of non-parasitized E in peripheral blood films of these patients (Fig. 3). Erythrophagocytosis by polymorphonuclear cells was never observed although malarial pigment was often seen in neutrophils and basophils. There was no association between the antiglobulin titre and anaemia (Table 3).
Close follow-up study Haematological and parasitological findings of the two children in this study are shown in Fig. 4. In patient JM, IgG appeared on the erythrocytes the day following chloroquine chemotherapy, followed by a decrease in PCV and Hb. However, both PCV and Hb returned to normal during the following week, although EIgG could still be demonstrated. The high initial parasitaemia of this patient might therefore explain the drop in PCV after chloroquine chemotherapy. Patient MH, with initial low paraTABLE 2. Specificity of DAT as related to anaemia in acute P. fakiparum malaria
Specificity of E sensitization (%o) IgG Fajara children Anaemic
C3d
+ C3d
0
69-2 (19)
15-3
15.3
(4)
(4)
8.3
75-0
16-6
0
(1)
(9)
(2)
(0)
(0) Not anaemic
IgG + C3d C3b
+ C4b +
IgG
Figures in parentheses are the number ofindividuals in each group. The child with EC4bC3bC3d was included in the last group.
123
Antiglobulin reactions in malaria
FIG. 3. Erythrophagocytosis by monocytes in the peripheral blood of a child with falciparum malaria and (Magnification x 2000.)
severe anaemia.
sitaemia, was anaemic when admitted but became severely anaemic following chloroquine chemotherapy, and the sharp drop in PCV and Hb coincided with the appearance of E-bound active C3 and C4. Likewise the disappearance of active C3 and C4 led to a marked recovery in PCV and Hb. Long-term fllow-up study Alterations in DAT specificity following chemotherapy (Fajara) and observations over a 6 week study period (Brefet) are shown in Tables 4 and 5. A number of differences were observed between the two groups. The majority of the patients with acute malaria who had a positive DAT on presentation, remained positive following chemotherapy, most retaining the same specificity (Table 4). A much lower percentage in the Brefet group followed this pattern (Table 5). A greater percentage of the Brefet children who had a negative DAT on presentation remained negative when compared to the Fajara group.
TABLE 3. DAT titres from children with acute P. fakiparum malaria with or without anaemia DAT log2 titre
Fajara children Anaemic Not anaemic
IgG
C3d
C3b
C4b
7-3±0-6 7-2±0-2
4-0±0-2
1-2±0-2 0
1-3±0-2
4 0±0 5
Log2 titres given as a meanl 1 s.e.
0
124
Christine A. Facer, R. 30
a-
a-
