Clin. exp. Immunol. (1992) 90, 357-362
Intravenous immunoglobulin in the treatment of paediatric cerebral malaria T. E. TAYLOR*, M. E. MOLYNEUXt, J. J. WIRIMAJ, A. BORGSTEIN, J. D. GOLDRINGt & M. HOMMELt Department of Paediatrics, Queen Elizabeth Central Hospital, Blantyre, Malawi, *Department of Community Health Sciences, College of Osteopathic Medicine, Michigan State University, MI, USA, tLiverpool School of Tropical Medicine, UK, and tCollege of Medicine, University of Malawi, Malawi
(Acceptedfor publication 9 September 1992)
SUMMARY Hyperimmune globulin can inhibit and reverse the cytoadherence between Plasmodiumfalciparuminfected erythrocytes and melanoma cells in vitro. Cytoadherence is believed to mediate disease in cerebral malaria. Therefore we studied the efficacy of i.v. immunoglobulin, purified from the plasma of local semi-immune blood donors, as an adjunct to standard treatment for cerebral malaria in Malawian children. The immunoglobulin preparation (IFAT antimalarial antibody titre 1:5120) recognized erythrocyte-associated antigens of each of 22 Malawian P.falciparum isolates studied, and reversed binding of Malawian isolates to melanoma cells. Immunoglobulin did not reverse binding to human monocytes or to cells of the human hystiocytic lymphoma cell line U937. Thirty-one children with P.falciparum parasitaemia and unrousable coma were enrolled. All were treated with i.v. quinine dihydrochloride; in addition patients were randomized to receive either immunoglobulin (400 mg/kg by i.v. infusion over 3 h) or placebo (albumen and sucrose by similar infusion) in a double blind trial with sequential analysis. Of 16 patients receiving immunoglobulin, five (31 %) died and five survivors had neurological sequelae. Of 15 patients receiving placebo, one (7%) died and two had sequelae. Parasite clearance, fever clearance and coma resolution times in survivors were similar in the two groups. Although the difference in outcome between the two groups was not significant, the trial was stopped because immunoglobulin was demonstrated not to be superior to placebo. Keywords cerebral malaria intravenous immunoglobulins immunotherapy randomized controlled trials
INTRODUCTION With the best available treatment the mortality of cerebral malaria (CM) in children in endemic areas is 20-33% [1,2]. This level of mortality is found even if parasites are fully sensitive to the antimalarial drug used [3], suggesting that in patients with CM, additional treatment directed at the pathogenetic processes responsible for disease may be beneficial. In falciparum malaria erythrocytes containing mature parasites adhere to microvascular endothelium and sequester in the venules and capillaries of deep tissues [4]. Sequestration of parasitized erythrocytes in the brain is believed to be important in the pathogenesis of CM [5]. Severe malaria is uncommon among semi-immune people living in endemic areas, although Plasmodium falciparum parasitaemia is common [2]. Much of this reduced susceptibility to severe disease may be the result of specific circulating antibody [6].
Immune serum can inhibit and reverse cytoadherence between P. falciparum-parasitized erythrocytes and a variety of cell lines cultured in vitro [7,8], and has been shown to dislodge sequestered parasitized erythrocytes in a squirrel monkey model of falciparum malaria [9]. These observations suggest that the administration of antimalarial immunoglobulin may, by reversing or preventing sequestration, be beneficial in severe malaria. In studies ofcytoadherence reversal, both in vitro and in vivo, the effect of immune serum has been shown to be parasite strainspecific [7,9]. In any human population there is considerable antigenic diversity among P.falciparum isolates [10]. Therefore, immune globulin for immunotherapy should ideally be prepared from the plasma of a large number of local adults, so that it is likely to contain antibody to most local strains of P.
falciparum. We have studied the effect of adding i.v. immune globulin, prepared from the pooled plasma of local blood donors, to standard therapy for cerebral malaria, in a double-blind placebo-controlled trial in Malawian children.
Correspondence: Malcolm E. Molyneux, School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK.
357
358
T. E. Tailor et al. PATIENTS AND METHODS
Because of the known prognostic importance of hypoglycaemia
Tle study, at the Queen Elizabeth Central Hospital. Blantyre,
[17,18], we decided in advance to stratify patients into normoand hypoglycaemic for each analysis.
Malaclwi, wals approved by the Nationall Health Sciences Research Committee of Malawi. Parents or guardians gave consent for the enrolment of each child, after the study had been explained to them In their own lanalguage. Preparation ol')fionunlitoglobhlin/rhoi i pooled plasicl Plasma for this study was obtained from blood donated for erythrocyte transfusions by 834 Malawian adults who were seronegative for anti-HIV antibodies (ELISA. Wellcome). Plasma was air-freighted on dry ice to the Central Laboratory of the Swiss Red Cross Blood Transfusion Service in Berne, Switzerland. Samples negative for both HBsAg (Ausria II, Abbott) and anti-H IV antibodies (anti H IV- 1, EIA, Abbott; anti HIV- I + 2, EIA, Abbott) were pooled and submitted to the comineicial process olfimmunoglobulin extraction. The process includes cold ethanol fractionation aind pH4 treatmnent, both procedures shown to remove and or inactivate viruses [11,12]. In particular these procedures have been demonstrated to inactivate over loi5 HIV in vitro infectious units (IVIU)/ml of plasma, the titre of HIV in the plasma of viraemic individuals being less than 102 IVI U /ml [ 1 3]. Epidemiological evidence also suggests that the risk of transmission of HIV by immunoglobulin prepared in this way is negligible [4]. The product. which met all the requirements of quality control, was issued in sealed rubber-topped glass vials, each containing 1 0 g immunoglobulin lyophilized in 18 gSucrose. Matching control vials contained a powder of identical appearance containing 1 8 g sucrose and 10 mg albumin. The immunoglobulin preparation consisted of 98"O IgG, of which 96"%i was monomeric and dimeric IgG, 3", IgG fragments and I IgG aggregates.
Design Of the studyl Mortality in CM is greatest among patients with profound coma [1]. In 86 patients previously seen with coma scores of 0 or I (no or undirected response to standard painful stimulus). 33", died or suffered neurological sequclac. The present study was designed to detect a reduction in the proportion of such adverse outcomes to 10"/%. In an initial open pilot study. four patients received immunoglobulin (50 mg, kg. 100 mgkg, 200nmgkg Lnd 400 mg, kg respectively). No adverse effect was observed, and the controlled trial was then commenced. The sequential analysis incorporated two alternative stopping rules. The trial would be stopped if the outcome proved to be significantly better in one group than in the other. The probability of detecting a statistically significant result, at a twosided significance level of 0 05, was set to be 0-9 if the proportion of failures on placebo was 033, compared with a proportion of failures of 0 1 on immunoglobulin (indicating a superiority of immunoglobulin). (The difference between treatments was considered in terms of the log odds-ratio.) Alternatively the trial would be stopped if outcomes in the immunoglobulin group could be shown to be no better (even if they were not significantly worse) than outcomes in the placebo group. Owing to the fact that seriously ill children were to be enrolled in the study, a sequential monitoring procedure was adopted [15], analyses being performed with PEST software [16]. We planned to monitor the data after 20 patients had completed treatment, then after every following 10 patients.
Patients were randomized to receive either placebo or immunoglobulin in addition to standard therapy. Randomization was by computer-generated random tables in blocks of 10, so that among every 10 patients studied, five would receive immunoglobulin and five placebo. Clinical investigators were unaware of the treatment allocation.
Patients and clinical methods Children aged between I and 12 years of age were enrolled in the study if profoundly unconscious (coma score 0/5 or 1/5 [1]), with asexual P. falciparum parasitaemia and no other identifiable cause of fever or coma. After initial clinical assessment all patients were treated with i.v. quinine dihydrochloride 20 mg/kg infused over 3 h, followed by 10 mg/kg every 8 h until oral treatment could be given (Fansidar®, 1-3 tablets as a single dose). Immunoglobulin or placebo was administered during the first 3 h through a side arm of the infusion set. General management of the patients was according to protocols described previously [1]. Pretreatment tests included thick and thin blood films for measurement of parasitaemia, packed cell volume (microhaematocrit centrifuge), haemoglobin and full blood count (Coulter counter), capillary pH and pCO2 (Radiometer ABL 330 blood gas analyser), capillary blood glucose concentration (BM-Test Glycemie 1--44 strips, Boehringer, read in a Medistron reflectance meter), serum complement (C3 and C4, rate nephelometry, Beckman Array), plasma lactate (YSI model 23 AM glucose analyser with L lactate oxidase membrane, Clandon Scientific) and plasma creatinine (Jaffe reaction). In all patients a lumbar puncture was done to exclude meningitis. Coma score and vital signs were assessed every 30 min for 6 h, and subsequently every 2 h until consciousness was regained. Before discharge from hospital each patient was assessed for the presence of neurological sequelae. Thick and thin blood films were repeated half-hourly for 6 h and then every 4 h during treatment until two successive films were negative. Capillary blood pH and pCO, were measured every 4 h until normal. Haematocrit was measured every 8 h. Serum complement measurements were repeated at 24 h. All patients who survived were reviewed I month after admission, when they were assessed clinically, blood films were taken and the haematocrit and serum complement were measured. Imnunofluorescent antibody, test (IFA T) The immunoglobulin preparation was assayed in an IFAT using acetone-fixed P. fidliparumn schizont-infected cells on 12-well 'toxoplasma' slides (Henley Essex Ltd). The titre of the reconstituted immunoglobulin preparation was 1:5120.
Binding of immunoglohulin
ervthroc'vtes The
to
sulface antigens of parasitized
immunoglobulin preparation was used in an immunogold/ silver enhancement technique, developed and routinely used in our laboratory [19] to assay the binding of antibody to erythrocyte-associated malaria surface antigen in 22 isolates obtained from children with severe malaria in Malawi. Antibody which remained bound to the surface of infected erythrocytes was then eluted, and its specificity tested by immunogold!
Treatment of paediatric cerebral malaria silver enhancement against homologous and heterologous isolates. Although the parasites tested were not taken from patients in the immunoglobulin trial (only a few of which could be cryopreserved), they were taken from comparable patients from the same geographical site, several being obtained during the period of the immunoglobulin trial.
Effects of immunoglobulin on cytoadherence in vitro Parasite isolates from patients with severe and mild malaria (not included in the immunoglobulin trial) were preserved in the spun deposit of the initial blood sample by step-wise cooling and storage in liquid nitrogen [20]. Isolates were subsequently retrieved and grown for studies of cytoadherence to monocytes, a human hystiocytic lymphoma cell line (U937) and melanoma cells. The effect of immunoglobulin on cytoadherence in vitro was assessed by inhibition and reversal assays [9].
Table 1. Pretreatment characteristics. Figures are means (s.d.) for continuous variables, and numbers (%) for categorical variables
Immunoglobulin-treated Placebo-treated (n = 16) Age (years) Sex, male/female Duration of fever (days) Duration of coma (days) History of convulsions Rectal temperature ( C)
Pulse rate (/mi)
Systoi103 (18)
(mmHg)rate/mmn Breathing
Liver (cm palpable) Spleen (cm palpable)
After 31 patients (16 of whom had received immunoglobulin, 15 placebo) had been enrolled, the trial was stopped because the sequential procedure showed that immunoglobulin was not superior to placebo. Data summaries for these patients are shown in Tables 1 and 2. The two groups proved to be closely matched in their demographic and pretreatment clinical features (Table 1). In particular, clinical components of the syndrome of CM that have been shown previously to be of prognostic importance-depth of coma, hypoglycaemia, witnessed convulsions, signs of decerebration/decortication, hyperparasitaemia [I] and acid-base status [21]-were similarly distributed between the two groups. One patient was undergoing treatment (i.e. had already received immunoglobulin) when the trial was stopped. This patient finished treatment according to protocol specifications, bringing the total number of patients in the final analysis to 32.
7/8 1-72 (1 7) 0-22 (0 21) 13 (87%)
38.7 (144)
38.9 (104)
148 (30) 103
152 (16) 101 (14) (14) 53 (14) 53 1 87 (2 02) 1-81 (1-92) 6 (40%) 5 (339%) 5 (33%) 3 (20'%) 2 (13%5)
(14)
Haematocrit, per cent
283 (826)
Convulsion observed
3 17 (1-7)
48 (14)
Hypoglycaemia
'Decerebration'*
(n = 15)
3-88 (2 0) 7/9 1 44 (1 09) 0 30 (0-28) 14 (88%)
1 25 (1 8) 1 59 (2-18) 11 (69%) 6 (38%) 11 (69%) 3 (19%) 3 (190%)
No. with coma score=0 Absent corneal reflexes
RESULTS
359
Blood leucocyte count ( x 109/l) 16-4 (9-9) Capillary blood pH 7 25 (0-12) Capillary blood pCO2 32-1 (15 9) (mmHg) Standard base excess - 12-4 (5 6) Plasma lactate (mmol/l) 7-36 (4-4) Plasma creatinine (timol/l) 61 5 (141)
2737 (636) 17-5 (13-3) 7 31 (0 10)
53
31-2 (7 4) -9.9 (4 8) 5-64 (4-5) 5 (23 7)
Statistical comparisons between the two groups of patients (MannWhitney tests for groups of continuous variables, or X2-test with Yates correction for categorical variables) showed no significant differences for any of the items listed. * Hypertonicity, opisthotonus or posturing.
Outcome Five patients were hypoglycaemic on entry to the study. Of these, 3/3 failed (died or had neurological sequelae) on immunoglobulin and 0/2 failed on placebo. Twenty-seven patients were normoglycaemic: in this sub-group the failure rate on immunoglobulin was 7/14, compared with 3/10 on placebo. These results were pooled and an analysis was performed which allowed for the sequential monitoring, yielding a two-sided significance of 0 09. The median unbiased estimate of treatment effect in terms of the odds ratio was 0-24 (95% CI 0-05, 1-26)-i.e. the odds of failing on placebo were approximately one-quarter of the odds of failing on immunoglobulin. Expressed differently, the probability of failing on treatment was estimated as 0-23 on placebo and 0 56 on immunoglobulin. At this point in the trial, the difference in outcomes between the two groups was sufficient to indicate that immunoglobulin could not be superior to placebo. The trial was therefore discontinued in accordance with the stopping rule. Numbers were insufficient to distinguish whether immunoglobulin was inferior or merely equivalent to placebo, but it was not an objective of the trial to prove or disprove inferiority of immunoglobulin. Therefore no further patients were enrolled. The mortality among patients receiving immunoglobulin (5/16) was similar to the overall mo~rtality rate of 17/86 observed
in previous studies for children with coma scores of 0-1 ([1] and unpublished data). In patients receiving immunoglobulin, deaths occurred at 5, 16, 21, 25 and 87 h (median 21 h) after the start of treatment. Death in a patient receiving placebo occurred at 3 h. In the 17 patients with CM and comparable depth of coma who died in previous studies, the median time of death was 9 5 h (range 1-31 h). Recovery rates. There were no significant differences, among survivors in the two treatment groups, in the rates of decrease of parasitaemia, resolution of fever and recovery of consciousness (Table 2). Clinical events during treatment. No differences in the pattern of clinical events or in the manner of death could be discerned between immunoglobulin recipients and placebo recipients, or between immunoglobulin recipients and patients studied previously. Serum complement levels before and after treatment. The mean serum C3 and C4 concentrations were significantly lower on admission to hospital than in convalescence (Table 3). There were no significant differences between immunoglobulin recipients and placebo recipients in their mean C3 or C4 levels at any of the sampling times. Mean initial serum C3 and C4
360
T. E. Taylor et al. Table 2. Outcome variables Placebo
Immunoglobulin-treated Number of patients
16
15
Died/sequelae/full recovery* Hours to full consciousness
5/5/6 23-0 (19) (n= 6)
1/2/12 22 6 (18) (n= 13)
Fever resolution (h)
41 5 (11) (n= 11) 40 5 (11) (n= 12)
46 6 (24) (n= 14)
Parasite clearance (h) 50% parasite clearance (h) 90% parasite clearance (h) Spleen enlargement on treatment
17 (9) (n= 13) 23 (8) (n= 13) 10 (59%)
34 9 (7) (n= 14) 15 (8) (n= 15) 20 (7) (n= 15) 5 (31%)
Differences between groups are non-significant for each variable (Mann-Whitney tests for groups of continuous variables, or X2-test with Yates correction for categorical variables). * P=0 08 (adjusted for sequential design).
Table 3. Mean (s.d.) serum complement concentrations (g/l) at various times in relation to start of treatment 24 h
f/u
P*
085 (041)
079 (031)
123 (022)
002
081 (035)
093 (027)
1 16 (021)
001
0h
Mean C3 Immunoglobulin Placebo
the 22 Malawi isolates studied [22]. The eluted antibody from each isolate was shown to recognize the homologous isolate only, and to have no affinity for heterologous isolates. These results indicate that the immunoglobulin preparation contains numerous antibodies with different specificities; it does not consist of a single or limited group of antibodies that cross-react between isolates.
Cytoadherence assays Melanoma cell binding assay (MCBA). Most P. falciparum isolates from Thailand bind readily to melanoma cells in vitro Mean C4 [23]. By contrast, few isolates from Malawi were found to adhere Immunoglobulin 0 18 (0 09) 0 20 (0-06) 0 25 (0 08) 0 02 to melanoma cells. The effect of immunoglobulin on the MCBA 0 16 (0 08) 0 23 (0 07) 0-22 (0 06) 0 04 Placebo 0 17 (0 09) 0 21 (0 06) 0 23 (0 07) 0 01 was studied with adhering isolates from both sources. Three Combined isolates from Thai patients and six isolates from patients in Malawi were studied. Immunoglobulin significantly prevented P= Significance of difference between 0 h and f/u f (Mann-Whitney (Mann-Whitney and reversed the binding of all isolates from both sources, At follow-up visit I month after treatment.adherence of the Malawian isolates being inhibited by a mean of 62% (range 50-87%) when compared with control assays using normal human immunoglobulin. concentrations were similar in patients who made a full recovery Binding to human monocytes and U937 cells. Plasmodium and those who died or suffered neurological sequelae. There was falciparum isolates from Malawi showed a greater degree of a negative correlation between the density of parasitaemia and binding to monocytes and U937 cells than to melanoma cells the serum concentration of C3 (r = - 0460, P
Intravenous immunoglobulin in the treatment of paediatric cerebral malaria T. E. TAYLOR*, M. E. MOLYNEUXt, J. J. WIRIMAJ, A. BORGSTEIN, J. D. GOLDRINGt & M. HOMMELt Department of Paediatrics, Queen Elizabeth Central Hospital, Blantyre, Malawi, *Department of Community Health Sciences, College of Osteopathic Medicine, Michigan State University, MI, USA, tLiverpool School of Tropical Medicine, UK, and tCollege of Medicine, University of Malawi, Malawi
(Acceptedfor publication 9 September 1992)
SUMMARY Hyperimmune globulin can inhibit and reverse the cytoadherence between Plasmodiumfalciparuminfected erythrocytes and melanoma cells in vitro. Cytoadherence is believed to mediate disease in cerebral malaria. Therefore we studied the efficacy of i.v. immunoglobulin, purified from the plasma of local semi-immune blood donors, as an adjunct to standard treatment for cerebral malaria in Malawian children. The immunoglobulin preparation (IFAT antimalarial antibody titre 1:5120) recognized erythrocyte-associated antigens of each of 22 Malawian P.falciparum isolates studied, and reversed binding of Malawian isolates to melanoma cells. Immunoglobulin did not reverse binding to human monocytes or to cells of the human hystiocytic lymphoma cell line U937. Thirty-one children with P.falciparum parasitaemia and unrousable coma were enrolled. All were treated with i.v. quinine dihydrochloride; in addition patients were randomized to receive either immunoglobulin (400 mg/kg by i.v. infusion over 3 h) or placebo (albumen and sucrose by similar infusion) in a double blind trial with sequential analysis. Of 16 patients receiving immunoglobulin, five (31 %) died and five survivors had neurological sequelae. Of 15 patients receiving placebo, one (7%) died and two had sequelae. Parasite clearance, fever clearance and coma resolution times in survivors were similar in the two groups. Although the difference in outcome between the two groups was not significant, the trial was stopped because immunoglobulin was demonstrated not to be superior to placebo. Keywords cerebral malaria intravenous immunoglobulins immunotherapy randomized controlled trials
INTRODUCTION With the best available treatment the mortality of cerebral malaria (CM) in children in endemic areas is 20-33% [1,2]. This level of mortality is found even if parasites are fully sensitive to the antimalarial drug used [3], suggesting that in patients with CM, additional treatment directed at the pathogenetic processes responsible for disease may be beneficial. In falciparum malaria erythrocytes containing mature parasites adhere to microvascular endothelium and sequester in the venules and capillaries of deep tissues [4]. Sequestration of parasitized erythrocytes in the brain is believed to be important in the pathogenesis of CM [5]. Severe malaria is uncommon among semi-immune people living in endemic areas, although Plasmodium falciparum parasitaemia is common [2]. Much of this reduced susceptibility to severe disease may be the result of specific circulating antibody [6].
Immune serum can inhibit and reverse cytoadherence between P. falciparum-parasitized erythrocytes and a variety of cell lines cultured in vitro [7,8], and has been shown to dislodge sequestered parasitized erythrocytes in a squirrel monkey model of falciparum malaria [9]. These observations suggest that the administration of antimalarial immunoglobulin may, by reversing or preventing sequestration, be beneficial in severe malaria. In studies ofcytoadherence reversal, both in vitro and in vivo, the effect of immune serum has been shown to be parasite strainspecific [7,9]. In any human population there is considerable antigenic diversity among P.falciparum isolates [10]. Therefore, immune globulin for immunotherapy should ideally be prepared from the plasma of a large number of local adults, so that it is likely to contain antibody to most local strains of P.
falciparum. We have studied the effect of adding i.v. immune globulin, prepared from the pooled plasma of local blood donors, to standard therapy for cerebral malaria, in a double-blind placebo-controlled trial in Malawian children.
Correspondence: Malcolm E. Molyneux, School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK.
357
358
T. E. Tailor et al. PATIENTS AND METHODS
Because of the known prognostic importance of hypoglycaemia
Tle study, at the Queen Elizabeth Central Hospital. Blantyre,
[17,18], we decided in advance to stratify patients into normoand hypoglycaemic for each analysis.
Malaclwi, wals approved by the Nationall Health Sciences Research Committee of Malawi. Parents or guardians gave consent for the enrolment of each child, after the study had been explained to them In their own lanalguage. Preparation ol')fionunlitoglobhlin/rhoi i pooled plasicl Plasma for this study was obtained from blood donated for erythrocyte transfusions by 834 Malawian adults who were seronegative for anti-HIV antibodies (ELISA. Wellcome). Plasma was air-freighted on dry ice to the Central Laboratory of the Swiss Red Cross Blood Transfusion Service in Berne, Switzerland. Samples negative for both HBsAg (Ausria II, Abbott) and anti-H IV antibodies (anti H IV- 1, EIA, Abbott; anti HIV- I + 2, EIA, Abbott) were pooled and submitted to the comineicial process olfimmunoglobulin extraction. The process includes cold ethanol fractionation aind pH4 treatmnent, both procedures shown to remove and or inactivate viruses [11,12]. In particular these procedures have been demonstrated to inactivate over loi5 HIV in vitro infectious units (IVIU)/ml of plasma, the titre of HIV in the plasma of viraemic individuals being less than 102 IVI U /ml [ 1 3]. Epidemiological evidence also suggests that the risk of transmission of HIV by immunoglobulin prepared in this way is negligible [4]. The product. which met all the requirements of quality control, was issued in sealed rubber-topped glass vials, each containing 1 0 g immunoglobulin lyophilized in 18 gSucrose. Matching control vials contained a powder of identical appearance containing 1 8 g sucrose and 10 mg albumin. The immunoglobulin preparation consisted of 98"O IgG, of which 96"%i was monomeric and dimeric IgG, 3", IgG fragments and I IgG aggregates.
Design Of the studyl Mortality in CM is greatest among patients with profound coma [1]. In 86 patients previously seen with coma scores of 0 or I (no or undirected response to standard painful stimulus). 33", died or suffered neurological sequclac. The present study was designed to detect a reduction in the proportion of such adverse outcomes to 10"/%. In an initial open pilot study. four patients received immunoglobulin (50 mg, kg. 100 mgkg, 200nmgkg Lnd 400 mg, kg respectively). No adverse effect was observed, and the controlled trial was then commenced. The sequential analysis incorporated two alternative stopping rules. The trial would be stopped if the outcome proved to be significantly better in one group than in the other. The probability of detecting a statistically significant result, at a twosided significance level of 0 05, was set to be 0-9 if the proportion of failures on placebo was 033, compared with a proportion of failures of 0 1 on immunoglobulin (indicating a superiority of immunoglobulin). (The difference between treatments was considered in terms of the log odds-ratio.) Alternatively the trial would be stopped if outcomes in the immunoglobulin group could be shown to be no better (even if they were not significantly worse) than outcomes in the placebo group. Owing to the fact that seriously ill children were to be enrolled in the study, a sequential monitoring procedure was adopted [15], analyses being performed with PEST software [16]. We planned to monitor the data after 20 patients had completed treatment, then after every following 10 patients.
Patients were randomized to receive either placebo or immunoglobulin in addition to standard therapy. Randomization was by computer-generated random tables in blocks of 10, so that among every 10 patients studied, five would receive immunoglobulin and five placebo. Clinical investigators were unaware of the treatment allocation.
Patients and clinical methods Children aged between I and 12 years of age were enrolled in the study if profoundly unconscious (coma score 0/5 or 1/5 [1]), with asexual P. falciparum parasitaemia and no other identifiable cause of fever or coma. After initial clinical assessment all patients were treated with i.v. quinine dihydrochloride 20 mg/kg infused over 3 h, followed by 10 mg/kg every 8 h until oral treatment could be given (Fansidar®, 1-3 tablets as a single dose). Immunoglobulin or placebo was administered during the first 3 h through a side arm of the infusion set. General management of the patients was according to protocols described previously [1]. Pretreatment tests included thick and thin blood films for measurement of parasitaemia, packed cell volume (microhaematocrit centrifuge), haemoglobin and full blood count (Coulter counter), capillary pH and pCO2 (Radiometer ABL 330 blood gas analyser), capillary blood glucose concentration (BM-Test Glycemie 1--44 strips, Boehringer, read in a Medistron reflectance meter), serum complement (C3 and C4, rate nephelometry, Beckman Array), plasma lactate (YSI model 23 AM glucose analyser with L lactate oxidase membrane, Clandon Scientific) and plasma creatinine (Jaffe reaction). In all patients a lumbar puncture was done to exclude meningitis. Coma score and vital signs were assessed every 30 min for 6 h, and subsequently every 2 h until consciousness was regained. Before discharge from hospital each patient was assessed for the presence of neurological sequelae. Thick and thin blood films were repeated half-hourly for 6 h and then every 4 h during treatment until two successive films were negative. Capillary blood pH and pCO, were measured every 4 h until normal. Haematocrit was measured every 8 h. Serum complement measurements were repeated at 24 h. All patients who survived were reviewed I month after admission, when they were assessed clinically, blood films were taken and the haematocrit and serum complement were measured. Imnunofluorescent antibody, test (IFA T) The immunoglobulin preparation was assayed in an IFAT using acetone-fixed P. fidliparumn schizont-infected cells on 12-well 'toxoplasma' slides (Henley Essex Ltd). The titre of the reconstituted immunoglobulin preparation was 1:5120.
Binding of immunoglohulin
ervthroc'vtes The
to
sulface antigens of parasitized
immunoglobulin preparation was used in an immunogold/ silver enhancement technique, developed and routinely used in our laboratory [19] to assay the binding of antibody to erythrocyte-associated malaria surface antigen in 22 isolates obtained from children with severe malaria in Malawi. Antibody which remained bound to the surface of infected erythrocytes was then eluted, and its specificity tested by immunogold!
Treatment of paediatric cerebral malaria silver enhancement against homologous and heterologous isolates. Although the parasites tested were not taken from patients in the immunoglobulin trial (only a few of which could be cryopreserved), they were taken from comparable patients from the same geographical site, several being obtained during the period of the immunoglobulin trial.
Effects of immunoglobulin on cytoadherence in vitro Parasite isolates from patients with severe and mild malaria (not included in the immunoglobulin trial) were preserved in the spun deposit of the initial blood sample by step-wise cooling and storage in liquid nitrogen [20]. Isolates were subsequently retrieved and grown for studies of cytoadherence to monocytes, a human hystiocytic lymphoma cell line (U937) and melanoma cells. The effect of immunoglobulin on cytoadherence in vitro was assessed by inhibition and reversal assays [9].
Table 1. Pretreatment characteristics. Figures are means (s.d.) for continuous variables, and numbers (%) for categorical variables
Immunoglobulin-treated Placebo-treated (n = 16) Age (years) Sex, male/female Duration of fever (days) Duration of coma (days) History of convulsions Rectal temperature ( C)
Pulse rate (/mi)
Systoi103 (18)
(mmHg)rate/mmn Breathing
Liver (cm palpable) Spleen (cm palpable)
After 31 patients (16 of whom had received immunoglobulin, 15 placebo) had been enrolled, the trial was stopped because the sequential procedure showed that immunoglobulin was not superior to placebo. Data summaries for these patients are shown in Tables 1 and 2. The two groups proved to be closely matched in their demographic and pretreatment clinical features (Table 1). In particular, clinical components of the syndrome of CM that have been shown previously to be of prognostic importance-depth of coma, hypoglycaemia, witnessed convulsions, signs of decerebration/decortication, hyperparasitaemia [I] and acid-base status [21]-were similarly distributed between the two groups. One patient was undergoing treatment (i.e. had already received immunoglobulin) when the trial was stopped. This patient finished treatment according to protocol specifications, bringing the total number of patients in the final analysis to 32.
7/8 1-72 (1 7) 0-22 (0 21) 13 (87%)
38.7 (144)
38.9 (104)
148 (30) 103
152 (16) 101 (14) (14) 53 (14) 53 1 87 (2 02) 1-81 (1-92) 6 (40%) 5 (339%) 5 (33%) 3 (20'%) 2 (13%5)
(14)
Haematocrit, per cent
283 (826)
Convulsion observed
3 17 (1-7)
48 (14)
Hypoglycaemia
'Decerebration'*
(n = 15)
3-88 (2 0) 7/9 1 44 (1 09) 0 30 (0-28) 14 (88%)
1 25 (1 8) 1 59 (2-18) 11 (69%) 6 (38%) 11 (69%) 3 (19%) 3 (190%)
No. with coma score=0 Absent corneal reflexes
RESULTS
359
Blood leucocyte count ( x 109/l) 16-4 (9-9) Capillary blood pH 7 25 (0-12) Capillary blood pCO2 32-1 (15 9) (mmHg) Standard base excess - 12-4 (5 6) Plasma lactate (mmol/l) 7-36 (4-4) Plasma creatinine (timol/l) 61 5 (141)
2737 (636) 17-5 (13-3) 7 31 (0 10)
53
31-2 (7 4) -9.9 (4 8) 5-64 (4-5) 5 (23 7)
Statistical comparisons between the two groups of patients (MannWhitney tests for groups of continuous variables, or X2-test with Yates correction for categorical variables) showed no significant differences for any of the items listed. * Hypertonicity, opisthotonus or posturing.
Outcome Five patients were hypoglycaemic on entry to the study. Of these, 3/3 failed (died or had neurological sequelae) on immunoglobulin and 0/2 failed on placebo. Twenty-seven patients were normoglycaemic: in this sub-group the failure rate on immunoglobulin was 7/14, compared with 3/10 on placebo. These results were pooled and an analysis was performed which allowed for the sequential monitoring, yielding a two-sided significance of 0 09. The median unbiased estimate of treatment effect in terms of the odds ratio was 0-24 (95% CI 0-05, 1-26)-i.e. the odds of failing on placebo were approximately one-quarter of the odds of failing on immunoglobulin. Expressed differently, the probability of failing on treatment was estimated as 0-23 on placebo and 0 56 on immunoglobulin. At this point in the trial, the difference in outcomes between the two groups was sufficient to indicate that immunoglobulin could not be superior to placebo. The trial was therefore discontinued in accordance with the stopping rule. Numbers were insufficient to distinguish whether immunoglobulin was inferior or merely equivalent to placebo, but it was not an objective of the trial to prove or disprove inferiority of immunoglobulin. Therefore no further patients were enrolled. The mortality among patients receiving immunoglobulin (5/16) was similar to the overall mo~rtality rate of 17/86 observed
in previous studies for children with coma scores of 0-1 ([1] and unpublished data). In patients receiving immunoglobulin, deaths occurred at 5, 16, 21, 25 and 87 h (median 21 h) after the start of treatment. Death in a patient receiving placebo occurred at 3 h. In the 17 patients with CM and comparable depth of coma who died in previous studies, the median time of death was 9 5 h (range 1-31 h). Recovery rates. There were no significant differences, among survivors in the two treatment groups, in the rates of decrease of parasitaemia, resolution of fever and recovery of consciousness (Table 2). Clinical events during treatment. No differences in the pattern of clinical events or in the manner of death could be discerned between immunoglobulin recipients and placebo recipients, or between immunoglobulin recipients and patients studied previously. Serum complement levels before and after treatment. The mean serum C3 and C4 concentrations were significantly lower on admission to hospital than in convalescence (Table 3). There were no significant differences between immunoglobulin recipients and placebo recipients in their mean C3 or C4 levels at any of the sampling times. Mean initial serum C3 and C4
360
T. E. Taylor et al. Table 2. Outcome variables Placebo
Immunoglobulin-treated Number of patients
16
15
Died/sequelae/full recovery* Hours to full consciousness
5/5/6 23-0 (19) (n= 6)
1/2/12 22 6 (18) (n= 13)
Fever resolution (h)
41 5 (11) (n= 11) 40 5 (11) (n= 12)
46 6 (24) (n= 14)
Parasite clearance (h) 50% parasite clearance (h) 90% parasite clearance (h) Spleen enlargement on treatment
17 (9) (n= 13) 23 (8) (n= 13) 10 (59%)
34 9 (7) (n= 14) 15 (8) (n= 15) 20 (7) (n= 15) 5 (31%)
Differences between groups are non-significant for each variable (Mann-Whitney tests for groups of continuous variables, or X2-test with Yates correction for categorical variables). * P=0 08 (adjusted for sequential design).
Table 3. Mean (s.d.) serum complement concentrations (g/l) at various times in relation to start of treatment 24 h
f/u
P*
085 (041)
079 (031)
123 (022)
002
081 (035)
093 (027)
1 16 (021)
001
0h
Mean C3 Immunoglobulin Placebo
the 22 Malawi isolates studied [22]. The eluted antibody from each isolate was shown to recognize the homologous isolate only, and to have no affinity for heterologous isolates. These results indicate that the immunoglobulin preparation contains numerous antibodies with different specificities; it does not consist of a single or limited group of antibodies that cross-react between isolates.
Cytoadherence assays Melanoma cell binding assay (MCBA). Most P. falciparum isolates from Thailand bind readily to melanoma cells in vitro Mean C4 [23]. By contrast, few isolates from Malawi were found to adhere Immunoglobulin 0 18 (0 09) 0 20 (0-06) 0 25 (0 08) 0 02 to melanoma cells. The effect of immunoglobulin on the MCBA 0 16 (0 08) 0 23 (0 07) 0-22 (0 06) 0 04 Placebo 0 17 (0 09) 0 21 (0 06) 0 23 (0 07) 0 01 was studied with adhering isolates from both sources. Three Combined isolates from Thai patients and six isolates from patients in Malawi were studied. Immunoglobulin significantly prevented P= Significance of difference between 0 h and f/u f (Mann-Whitney (Mann-Whitney and reversed the binding of all isolates from both sources, At follow-up visit I month after treatment.adherence of the Malawian isolates being inhibited by a mean of 62% (range 50-87%) when compared with control assays using normal human immunoglobulin. concentrations were similar in patients who made a full recovery Binding to human monocytes and U937 cells. Plasmodium and those who died or suffered neurological sequelae. There was falciparum isolates from Malawi showed a greater degree of a negative correlation between the density of parasitaemia and binding to monocytes and U937 cells than to melanoma cells the serum concentration of C3 (r = - 0460, P
