Studies on various aspects of the indirect haemagglutination test for malaria*

Bull. Org. mond. Santh 1972, 46, 771-782 Bull. Wid Hith Org. Studies on various aspects of the indirect haemagglutination test for malaria* J. H. E. T. MEUWISSEN, ANNA D. E. M. LEEUWENBERG, & G. E. MOLENKAMP Thispaper gives the results of studies on various technical aspects of the indirect haemag- glutination (IHA) test for malaria, on the similarity of the results obtained in the IHA test and in the indirect fluorescent antibody test, on the use of various plasmodial extracts as sensitizing antigens in the IHA test, and on the influence of heterophile antibodies on the titres obtained in the IHA test. Some longitudinal observations on induced malaria in- fections of man and monkey showed that the infection can induce the production of hetero- phile antibodies: their appearance, however, remains unpredictable. In some infections agglutinins against host erythrocyte components are also produced. Absorption of sera with tanned sheep cells sensitized with noninfected host red blood cell antigens is advocated as a control on the IHA titre for specific agglutinins. The indirect haemagglutination (IHA) test, so successfully applied in various fields of microbio- logy and immunology, still awaits wide acceptance as a routine method for the seroepidemiological study of malaria despite the fact that it was intro- duced for that purpose in 1962 by Desowitz & Stein (1962). It is obvious that a simple reproducible serological test for epidemiological studies in the field would be of great practical importance. Such a test could be applied to the assessment of malaria ende- micity, to the study of fluctuations in the intensity of malaria transmission, and to the evaluation of malaria control and eradication measures. For some years we have from time to time used the IHA technique in the course of experimental studies of malaria. This paper presents a preliminary report on our experience with the IHA test with particular attention to attempts to assess the influence on the test of the development of heterophile antibodies. It is hoped that the paper may stimulate other workers to explore ways of further improving the test. MATERIALS AND METHODS Sera Human serum samples collected from 276 people and sent to the laboratory between 1965 and 1970 * From the Department of Medical Microbiology, Uni- versity of Nijmegen, Nijmegen, The Netherlands. for examination by the indirect fluorescent antibody (IFA) test for malaria were also used for this study on haemagglutinins. Most of the sera were from Caucasians with symptoms of fever or splenomegaly, who had attended the outpatient department of a tropical diseases institute in the Netherlands, or from those having a routine medical examination following a stay in the tropics. Serum samples from four neurosyphilitic patients, blood-inoculated with Plasmodium ovale (Meuwissen, 1966), were also ex- amined by the IHA test.' Sera collected at regular intervals from rhesus monkeys inoculated between 1968 and 1970 with various species and strains of plasmodia were also used. All sera had been stored at - 20°C without preservatives. Parasites The following strains and species of malaria para- sites were used: Plasmodium cynomolgi bastianellii, P. c. langur, P. c. ceylonensis, P. inui, P. knowlesi, P. knowlesi Guy strain, P. fieldi, and P. vinckei. P. fieldi was obtained from Dr W. Collins (Chamblee, Ga., USA) and P. vinckei from the late Professor Vincke (Antwerp, Belgium); the other species of plasmodia were received from Dr A. Voller (London, England). 1 Some samples in the longitudinal follow-up study had been used in other experiments and were no longer available for examination by the IHA test. 2856 - 771- J. H. E. T. MEUWISSEN ET AL. Preparation of sensitive antigen Splenectomized rhesus monkeys were infected by intravenous inoculation with the primate plasmodia. The animals were bled at the moment of maximum parasite density, when most of the parasites were schizonts. The blood was defibrinated with glass beads and the cells were washed in phosphate buffered saline (PBS), pH 7.2, and resuspended in the original volume of PBS. To eliminate the white blood cells the erythrocytes were twice aggregated by addition of an equal volume of 3% dextran in PBS. To concentrate the parasitized cells, the erythrocytes were suspended in at least 3 volumes of PBS and centrifugated for 15 min at 1 100 g at 4°C. The lighter parasitized cells tended to form a darker band on top of the other erythrocytes. This band was carefully removed with a bent pipette. Following repeated separation of the bands through washing and centrifugation the remaining cells were resus- pended in PBS, the suspension was centrifuged, and the darker bands were combined. In this way a suspension containing mostly parasitized cells was prepared. These packed parasitized erythrocytes were stored at - 70°C in volumes of 0.1 ml. On the day of the actual test, 0.9 ml of PBS, pH 5.5, was added to the cells and the suspension was subjected to ultrasonic treatment for exactly 10 seconds, with a Branson Sonifier, model J-17V, equipped with a microtip and with the activity level control indicator at position 10 on the scale. After the ultrasonic treatment the suspension was centrifuged at 8 500 g for 15 min at 4°C. The supematant was the undiluted sensitizing antigen. P. vinckei was inoculated intraperitoneally into Swiss mice. Heavily infected blood from these ani- mals was collected in acid citrate dextrose solution and the sensitizing antigen was prepared as described above. Preparation of sensitized cells Sheep erythrocytes collected in Alsever solution and stored at 4°C were used within 8 days. As re- quired 0.3-ml quantities of packed thrice-washed cells were resuspended in 9.7 ml of 0.0025% tannic acid solution in PBS, pH 7.2, incubated at 4°C for 15 min, and washed several times, with a final wash- ing in PBS, pH 5.5. For antigen titrations, volumes of 0.06 ml of tanned cells were suspended in PBS, pH 5.5, up to a final volume of 1 ml after the addition of increasing quantities of antigen-namely, 0.06, 0.1, 0.2, and 0.3 ml-in different tubes. In a sixth tube, control cells were prepared without the addition of anti- gen. After incubation at 37°C for 30 min the cells were washed twice in a diluent, i.e. 1 % inactivated rabbit serum in PBS, pH 7.2. Suspensions of 1.3% sensitized cells and nonsensitized control cells were prepared: 0.025 ml of these suspensions were added to equal volumes of sera, inactivated at 56°C for 30 min, and diluted in the microtitre system (Sever, 1962) in a diluent, starting at a dilution of 1: 40. The microtitre plates were shaken mechanically for 2 min. The test was read after incubation for 1 h at room temperature and after storage overnight in a refrigerator. For the optimum quantity of sensitizing antigen, we used the smallest quantity that showed the highest haemagglutinin titre with a set of positive reference serum samples, in the absence of haemagglutination with sensitizied cells combined with a negative reference serum, and with nonsensi- tized cells combined with the positive references. Grades of agglutination were read according to Stavitsky (1954). A grade ± was considered posi- tive. Indirect haemagglutination test (IHA test) For the actual test, the optimum quantity of sensi- tizing antigen was added to tanned cells as determined in the antigen titration test. In some tests, tanned cells were sensitized with the constituents of non- infected erythrocytes prepared from noninfected blood. This " control antigen ' was prepared from noninfected blood in the same way as were the specific antigens from parasitized erythrocytes. All serological samples were inactivated at 56°C for 30 min. As a routine, they were tested with speci- fically sensitized cells and with nonsensitized tanned sheep cells as a control on the presence of hetero- phile antibodies, i.e., antibodies reacting with anti- genic determinants of noninfected heterologous erythrocytes. Absorption procedure. In some tests, serum samples were examined after absorption. Inactivated serum, diluted 1: 5, was mixed with 4 volumes ofa 25% suspension oferythro- cytes. In most experiments fresh sheep cells were used, but in others tanned sheep cells sensitized with " control antigen " were employed. After incubation at room temperature for 1 h the 1: 20 diluted supernatant serum sample was used in the IHA test. 772 INDIRECT HAEMAGGLUTINATION TE ST FOR MALARIA Table 1. IHA titres obtained with P. vinckei as sensitizing antigen. Influence of the duration of ultrasonic treatment on P. vinckei antigen (lot no. 96.02.14) tested in duplicate with mouse serum (A 11 F 6) IHA titres after ultrasonic treatment for the following no. of seconds: 10 20 40 60 80 100 120 140 first test 320 320 320 320 160 80 80 40 second test 640 320 80 160 160 160 80 40 RESULTS Technical factors that influenced the reproducibility of the test The preparation of antigen. The duration of the ultrasonic treatment appeared to be critical for the disruption of the parasitized cells. Treatment at the indicated level of energy output for less than 10 seconds did not result in complete disruption of all cells: more than 10 seconds however, reduced the antigenicity of the crude antigen (see Table 1). Repeated freezing and thawing also had a detri- mental effect on the antigenicity. Crude antigen stored at - 70°C did not lose its antigenicity within 20 weeks of storage but at - 20°C the antigen was much less stable. The pH during sensitization of the tanned cells. In our experience, the cells spontaneously agglutinate when the tanned cells are sensitized at a pH that approaches neutrality. The concentration of sensitized cells. One source of variability in the test appeared to be the concen- tration of the sensitized cells added to the diluted serum samples. At low concentrations the aggluti- nation patterns are difficult to read and at higher concentrations the titres tend to decrease. Source of cells. Table 2 shows the results of tests on different days with 1 negative and 4 positive sera that were absorbed with fresh erythrocytes. In these tests 6 different sheep were used as sources of cells. The cells were sensitized with the same batch of antigen. The table demonstrates that good reproduci- bility can be achieved when the same source of cells is used and that greater variability results from the use of erythrocytes from different sheep. Source ofantigen. The results of tests with 8 human sera that were examined with cells of 1 sheep sensi- tized with 3 batches of P. fieldi antigen showed that different batches of antigen can be a source of variability in the test. Comparison of titres obtained with IHA test and the IFA test Comparison in tests ofhuman sera. Table 3 shows the relationship between the IHA titres obtained with tanned cells sensitized with P. c. bastianellii antigen, and the titres obtained in the IFA test with P. fieldi antigen. Of the 276 sera tested, 233 had an IHA titre 61: 40; 215 of these 233 also had an IFA titre <1:40, indicating that in the low range of antibody titres both tests gave similar results in 92% of the tests. At the upper end of the scale the antibody titres were similar in about 70% of cases, 30 sera with IHA titres >1 : 160 having IFA titres >1: 80, although the IFA titres tended to be somewhat lower than the IHA titres. However, 18 (44%) of 44 sera with an IFA titre >1: 80 had an IHAltitre <1: 40 and 17 (40%) of 43 sera with an IHA titre A1: 80 had an IFA titre < 1: 40. Seven of the latter 17 sera, however, contained heterophile haemagglutinins: after they had been absorbed, the IHA titres became <1 :40. Comparison in a longitudinal study of monkey malaria. For a better comparison of the IHA and IFA tests a longitudinal study was made of five malaria infections in monkeys: one sporozoite- induced infection (D24) and four blood-induced infections with P. cynomolgi bastianellii, P. c. langur, P. knowlesi, and P. inui. In these five monkeys the heterophile antibody titre remained < 1: 40 during the whole observation period of 80 days after the first day of parasitaemia. In these sera the develop- ment of the IHA titres and of the IFA titres closely paralleled each other. The IHA antibody titres were higher in most ani- mals than the IFA titres. However, this difference 773 774 J. H. E. T. MEUWISSEN ET AL. Table 2. Indirect haemagglutination test for malaria with one batch of P. c. bastianelliiantigen. Titres obtained on different days with four positive and one negative test serum, absorbed with fresh sheep cells, in tests with sensitized (Sens.) and nonsensitized (Non) tanned cells: six different sheep cell donors were used Sheep no. Frequency Serum 18 12 9 1 13 16 of observations of -______________________vario us titres Sens. Non Sens. Non Sens. Non Sens. Non Sens. Non Sens. Non 190 1 280 <40 1 280 1 280 <40 640 1 280 640 640 <40 <40 <40 <40 <40 5120 5 120 1 280 1 280 (SP) (SP) <40 <40 640 <40 640 <40 105 1 280 <40 640 <40 640 <40 244 160 <40 640 640 <20 640 320 320 320 <40 <40 <40 <40 <40 2 at 5 120 6 at 1 280 6 at 640 320 <40 1 at 1 280 2 at 640 1 at 320 640 <40 320 <40 160 <40 640 <40 160 <40 276 320 <40 320 <40 640 <40 320 <40 640 <40 320 320 160 <40 <40 <40 640 320 <40 <40 5 at 640 4 at 320 3 at 160 3 at 640 7 at 320 1 at 160 320 <40 negative <40 <40 <40 serum <40 <40 <40 SP <40 <40 <40 <40 <40 <40 <40 SP SP <40 <40 <40 <40 <40 <40 <40 <40 <40 <40 <40 <40 could have resulted partly from the fact that P. cyno- molgi antigen was used in the IHA test whereas P. fieldi antigen was used in the IFA test. This is in agreement with the observation that the difference between IHA and IFAtitre values tended to be greater in two animals that received a homologous inocula- tion with P. cynomolgi bastianellii. IHA tests with various sensitizing antigens In order to demonstrate differences in reactivity, antigens prepared from P. cynomolgi bastianellii, P. c. langur, P. ceylonensis, P. inui, P. fieldi, P. know- lesi, and P. knowlesi Guy strain were compared in IHA tests with sera from persons with proved malaria parasitaemia. None of the sera used in this experi- ment agglutinated nonsensitized tanned sheep cells. A control antigen prepared from noninfected rhesus monkey erythrocytes was added to the series of plasmodial antigens. Table 4 shows the mean anti- body titres of 10 sera from persons with a P. falci- parum infection, of 14 sera from persons with P. vi- vax infections, of 3 sera from persons with P. malariae 3 SP 11 at <40 INDIRECT HAEMAGGLUTINATION TEST FOR MALARIA Table 3. Distribution of antibody titres in the IHA test of 276 human sera examined with tanned cells sensitized with P. c. bastianelli antigen according to the antibody titre in the IFA test with P. fieldi antigen IHA I FA titre titre <20 20 40 80 160 320 640 1 280 Total <40 101 56 39 1 2 1 209 40 1 3 3 3 4 1 24 80 4 3 4 3 2 1 17 160 1 3 5 1 10 320 4 0 0 1 1 6 640 1 1 1 1 4 1 280 3 0 1 4 2561 0 0 0 0 0 0 0 0 0 >5120 1 0 0 1 2 total 119 63 50 29 9 3 2 1 276 Table 4. Comparison of the mean titres obtained with seven sensitizing plasmodial antigens in the IHA test for malaria with sera negative for heterophile antibodies, and obtained from subjects with a natural malaria infection Antigen Infection No.of sera P. C. P. C. P. C.c P ni P ili P nwei P. knowlesi Controlbastianelli langur ceylonensis P. iGui P. fildi P. knowlesi Guy strain antigen P. falciparum 10 10 x 24.1 10 x 2 3-1 10 x 2 3o0 10 x 24.7 10 x 2 2.8 10 x 22-2 10 x 2 2-5 10 x 2 1.6 P. vivax 14 10 x 2 5'1 10 x 2 3-6 10 x 2 3-5 10 x 24-9 10 x 2 3.4 10 x 2 3.1 10 x 2 2.4 10x 2 1-2 P. malariae 3 10 x 26-0 10 x 24X3 10 x 24X3 10 x 25.3 10 x 24-7 10 x 24.7 10 x 240 10 x 21.3 P. ovale 2 10 x 25.0 10 x 23.5 10 x 23.5 10 x 24.5 10 x 22.5 10 x 22.5 10 x 22.5 10 x 21'5 infections, and of 2 sera from persons with P. ovale infections. Comparisons should be made only be- tween the titres of the sera against the different monkey malaria antigens and not between the results of the different sera. It is apparent that, whatever the source of the malaria serum samples, the IHA titres were highest in tests with P. cynomolgi and P. inui as sensitizing antigen and were lowest in those with P. knowlesi antigen. No category of sera had a particularly greater reactivity with any one of the sensitizing antigens. The sera examined in this experiment,with the exception of two sera from the P. fakiparum group, did not show at a dilution of 1: 40 haemagglutination with tanned cells sensitized with control antigen. Studies on the frequency ofaspecific positive reactions in the IHA test for malaria Examination of nonmalarious human sera. In this experiment we studied sera that could have been positive for aspecific reactions in the IHA test for malaria. For these tests tanned cells sensitized with P. c. bastianellii antigen were used. As indicated in Table 5, 19 (10%) of 189 sera had a titre >1: 40 in the IHA test. After absorption with fresh sheep cells, however, only 5 (3%) were still reactive in this test. Examination of bovine sera from animals infected with Anaplasma marginale. Sera from Nigerian cattle infected with Anaplasma marginale were tested using 775 J. H. E. T. MEUWISSEN ET AL. Table 5. IHA antibody titres of nonmalarious human sera before and after absorption with fresh sheep cells in sera with a titre > 1: 40 for tanned cells sensitized with P. c. bastianelli antigen IHA titre IHA titre Sera from people with: before absorption after absorption <1 :40 >1 :40 <1 :40 1:40 >1:80 clinically suspected rheumatoid arthritis: titre in the Rose- Waaler test >1 :64 32 4 3 1 1: 32 98 4 3 1 positive Wassermann test 2 1 1 Reiter's disease 0 1 1 mononucleosis 0 4 4 periarteritis nodosa 1 0 disseminated lupus erythematosus 1 3 3 hepatotenticular degeneration (Morbus Wilson) 1 0 carcinosis 1 0 dysglobulinaemia 2 0 pancytopenia 1 0 idiopathic haemolytic anaemia 1 0 IgG paraproteinaemia 5 0 increased IgM level of unknown origin 0 2 1 1 leukaemia 13 0 myelomatosis 12 0 total 170 19 14 4 1 tanned cells sensitized with P. c. bastianellii antigen. Without absorption with fresh sheep cells, 6 of 8 sera had positive titres between 1: 80 and 1: 1280. After absorption all titres were 1: 40. Development of heterophile antibodies during malaria infections and their influence on the IHA test During some parasitaemias in man and animals we observed the development of antibodies against unsensitized tanned sheep cells. P. ovale infections. The development of hetero- phile antibodies in a blood-induced P. ovale in- fection in patient D may be used as an example (Meuwissen, 1966). Prior to the inoculation with P. ovale, this patient had received several inoculations with P. vivax. From the sixth day of parasitaemia onwards antibodies against nonsensitized tanned sheep cells were present. Haemagglutinins against tanned sheep cells sensitized with P. c. bastianellii antigen appeared before this. The development of heterophile antibodies was observed in the sera of 2 of the 4 patients with P. ovale; in both of them one or more induced infections with P. vivax had preceded the inoculation with P. ovale. Monkey malaria infections. In 5 of 10 non- splenectomized rhesus monkeys, one sporozoite- induced and four blood-induced malaria infections were accompanied by the development of haemag- glutinins against sensitized tanned sheep cells and of heterophile antibodies. In three monkeys low heterophile antibody titres were present before the inoculation. In 3 of the animals the highest titre 776 INDIRECT HAEMAGGLUTINATION TEST FOR MALARIA Table 6. Distribution of IHA antibody titres of 276 sera from individuals tested with tanned cells sensitized with P. c. bastianellii antigen according to the IHA titre with nonsensitized cells IHA titre with sensitized IHA titre with nonsensitized tanned cells tanned cells <40 40 80 >1 60 Total <40 208 1 0 0 209 40 13 11 0 0 24 80 9 2 6 0 17 160 9 0 0 1 10 320 5 1 0 0 6 640 3 0 1 0 4 1 280 3 0 0 1 4 2 560 0 0 0 0 0 5120 1 0 0 1 2 total 251 15 7 3 276 of heterophile antibodies was seen after a second rise in parasite density. In all 5 animals the haemaggluti- nin titres for sensitized cells started to rise within a few days of the appearance of circulating parasites. Heterophile antibodies in human sera. The develop- ment of heterophile antibodies in natural malaria infections was further studied in an examination of the 276 human sera (see earlier section). Table 6 shows that 13 (30 Y.) of 43 sera with an IHA titre > 1: 80 with cells sensitized with P. bastianellii anti- gen contained heterophile antibodies with titres >1:40. Further analysis of circulating haemagglutinins Since the origin and specificity of the antigenic determinants reacting in the IHA test with circulating haemagglutinins is unknown, a further analysis was carried out with three types of tanned cell: (1) cells sensitized with P. c. bastianellii antigen, (2) non- sensitized tanned cells, and (3) cells sensitized with control antigen Absorption experiments with fresh sheep cells. Of the 276 human sera, the 50 with IHA titres > 1: 80 in tests with cells sensitized with P. bastianellii anti- gen were tested again before and after absorption with fresh sheep cells. Before absorption, 32 (64%) of the sera reacted only with the specifically sensitized cells. Two sera that reacted with specifically sensi- tized cells and with nonsensitized cells lost all reacti- vity during the absorption. Five sera that reacted with specifically sensitized cells and with cells sensi- tized with control antigen lost not only their hetero- phile reactivity but also a great part of their haemag- glutinin reactivity with specifically sensitized cells. Of the 11 sera with reactivity for all three types of cells, only 3 retained specific haemagglutinin reacti- vity but in one of them the absorption was not completely effective. Absorption with tanned sheep cells sensitized with control antigen. Of a similar series of 34 human and primate sera with IHA titres >1: 80 in tests with specifically sensitized cells, 14 also reacted with non- sensitized cells, with cells sensitized with control antigen, or with both. These 14 sera can be arranged in four groups according to the results of tests after absorption with fresh sheep cells or with tanned cells sensitized with control antigen. Table 7 shows that the 5 sera in group A reacted with all 3 types of cell. Their heterophile antibody activity could be absorbed as well by the tanned cells sensitized with control antigen as by the fresh sheep cells, without a complete loss of haemagglutinins for specifically sensitized cells in 4 of the 5 sera. In the 4 sera of group B and the 2 of group C, fresh sheep cells failed to absorb heterophile antibodies. However, they could be absorbed by tanned cells sensitized with 7 777 J. H. EL T. MEUWISSEN ET AL. Table 7. IHA titres of human and primate malarious sera in tests with tanned cells sensitized with P. c. bastianellii antigen (1), with nonsensitized tanned cells (2), and with tanned cells sensitized with control antigen (3). The tests were carried out before and after absorption either with fresh sheep cells or with tanned cells sensitized with control antigen IHA antibody titre Serum no. Before absorption After absorption After absorption with tanned cellssensitized with control antigen (1) (2) (3) (1) (2) (3) (1) (2) (3) group A SM 1 160 40 40 80 <40 <40 80 <40 <40 SM 27/2 2 560 40 80 1 280 <40 <40 1 280 <40 <40 F 33/2 80 40 40 80 <40 <40 80 <40 <40 E 40/12 80 40 <40 40 <40 <40 <40 <40 <40 SM 194 160 >160 160 <80 <80 <80 40 <40 <40 group B SM D6 160 80 <40 80 80 <40 160 <40 <40 SM D7 320 40 <40 320 40 <40 320 <40 <40 E 78/4 1 280 80 80 1 280 80 80 1 280 <40 <40 C 10/19 80 80 40 80 80 <40 <40 <40 <40 group C SM 85 40 40 160 80 <80 160 80 <40 <40 SM 229 160 160 160 <40 <40 80 <40 <40 <40 group D SM 39-2 20480 <40 >640 40 960 <80 640 20480 <40 320 SM 39/3 20480 <40 >640 40960 <80 >1 280 20480 <40 640 control antigen. Four of these 6 sera did not show an appreciable loss in IHA titre for specifically sensitized cells. It was remarkable that in the group B sera, fresh sheep cells failed to absorb heterophiles that reacted with nonsensitized tanned sheep cells. For the group D sera the results of absorption experi- ments carried out on two serum samples of a Moroc- can patient with a falciparum parasitaemia are shown. The heterophile antibody activity of these sera could not be absorbed either with fresh sheep cells or with the cells sensitized with control antigen. DISCUSSION Technical aspects Following the introduction of the IHA tes, for serological studies of malaria (Desowitz & Stein, 1962; Stein & Desowitz, 1964) four other centres have reported on their results with this technique (Bray, 1965; Bray & El-Nahal, 1966; Mahoney et al., 1966; Rogers et al., 1968; Wellde et al., 1969). The techniques applied in the previous studies differ from each other in several respects. The details of the various techniques used in the other five studies as well as our own are summarized in Table 8. It will be seen that our technique combined points from the other methods. In the present study blood was collected in a flask with glass beads for defibrination and for the elimination of thrombocytes and most of the polymorphonuclear leucocytes; the lympho- cytes and monocytes were then separated from the red blood cells by agglutination of the latter with dextran solution. The object of this procedure was to concentrate the parasitized erythrocytes. No attempt was made to isolate the parasite from its host erythrocyte, the maximum amount of antigenic 778 INDIRECT HAEMAGGLUTINATION TEST FOR MALARIA Table 8. Comparison of techniques used in six studies of the indirect haemagglutination test for malaria a Study no. Techniques 1 2 3 4 5 6 Purification of antigen elimination of thrombocytes elimination of polynuclear leucocytes elimination of lymphocytes/ monocytes with dextran Isolation of parasites from infected red cells applied with distilled water applied by treatment with saponin Homogenization of parasitized material by freezing and thawing treatment in French pressure cell + + + + + + + + + + + + ++ + tissue grinder Chromatographic separation Carrier: formalinised red cell tanned red cell Temperature during treatment with tannic acid Strength of tannic acid solution Source of carrier cell Temperature during sensitization Period of sensitization pH during sensitization Serum inactivated Absorbed with fresh sheep cells Diluent b + 37 C 1/20 000 sheep room temp. 15 min 7.2 0.125 % BSA + 37 C 1/20 000 sheep 40 C 15 min 7.2 1 % RS sheep 37 C 15 min 5.6 4 °C 1/20 000 humanC 37 °C 15 min 7.2 0.4 % RS 1 % RS a 1. Study by Stein & Desowitz (1964) 2. Studies by Bray (1965) and Bray & El Nahal (1966) 3. Study by Mahoney et al. (1966) 4. Study by Rogers et al. (1 968) 5. Study by Welide et al. (1965) 6. Present study. b BSA = bovine serum albumin RS = rabbit serum. c Group 0 blood. material was desired. A simple ultrasonic treatment (unpublished data). The main drawback of the chick- was suitable for homogenization of the parasitized en cells was that the chickens varied considerably in cells. their suitability as cell donors. Even an individual In our tests sheep cells were used after tests with chicken showed day-to-day variation in this respect. tanned chicken cells and a few tests using human Moreover the quantity of blood that could be ob- blood group 0 erythrocytes had given poor results tained from an individual chicken was rather small. 779 4C 1/10 000 sheep room temp. 30 min 5.6 1 % RS 4 C 1/40 000 sheep 37 C 15 min 5.5 (+) 1 % RS J. H. E. T. MEUWISSEN ET AL. Our studies confirm that antigenic material is present in the supernatant of disrupted parasitized cells and that it is thermolabile. Storage at temperatures above - 70°C, and long ultrasonic treatment, lessen- ed the antigenicity of the cell extract. As reported by Mahoney et al (1965), we found it better to sensitize the tanned cells at pH 5.5 instead of at pH 7.2. With regard to the sensitivity of the test it appears that the concentration of the sensitized cell suspension used in the microtitre plates had some influence on the outcome of the test, a high concentration tending to decrease the average titre. The reproducibility of the test depended partly on differences in carrier cells from different donor sheep. Since little is known about the nature of the sensi- tizing antigen, the malaria antigen content of a para- sitized cell extract cannot be assessed. As was to be expected, different batches of one species of antigen showed differences in reactivity with the same lot of sera. The sensitizing antigen may be either purely of parasite origin or it may be material composed of host and parasite components or even host cell material that has been modified by the presence of the parasite. In view of this uncertainty it appears probable that difficulties with the pre- paration of the sensitizing antigen will remain as long as there is no reliable measure of the specific antigen as content of the mixture of parasitized cell material. The other possibility has already been indicated by Weilde et al. (1969) in studies on chromatographic separation of the specific antigens; their observations should be confirmed and their experimental approach should be used more extensively by others. Comparison of the IHA and IFA tests The comparison of results obtained by applying the IHA and IFA techniques to the same sera con- firms in general the observations of Sadun et al. (1969) and Wilson et al. (1971). In their studies it was found that both tests provided a specific and sensitive method of following the course of antibody develop- ment in malaria infections. In the present study, 276 sera were tested with both methods. These sera were arbitrarily selected from individuals who had complained of symptoms that could be related to a possible malaria infection. In most cases parasi- taemia had not been demonstrated and only 43% of these sera showed an IFA titre >1: 20, indicating that only a minority of the subjects tested had ex- perienced a recent malaria attack. The data show that sera with low antibody contents give comparable titres with both methods and that in human sera with seropositive reactions, the titres in the IHA tests with P. c. bastianellii antigen tend to be higher in general than those in the IFA test with P. fieldi antigen. The longitudinal studies of monkey malaria seem to confirm these observations. They also show that in IHA reactions with homologous antigen the differences between titres in this test with P. cyno- molgi antigen and the IFA titres obtained with P.fieldi antigen tend to be greater than those obtained with heterologous antigens in both serological tests Comparison of different sensitizing antigens In accordance with current theories (WHO Scien- tific Group on the Immunology of Malaria 1968), our observations with the use of 7 different sensi- tizing antigens indicate that the IHA test for malaria demonstrates group-specific antibodies. In the study of human sera, only heterologous antigens were used. P. c. bastianellii and P. inui antigens proved to be the best sensitizing antigens for all tests, per- haps because of the developmental stage of the cir- culating parasites that were used for the preparation of the antigen. It seems that schizonts provide better antigens than young trophozoites. This could also be a possible explanation for the relatively poor antigenicity of the antigens prepared from P. know- lesi, the primate plasmodium with a unique 24-hour cycle. It is probable that with this species the anti- gen was not prepared within the short period when the greatest number of schizonts were circulating. Specificity of the test and influence of heterophile antibodies As was shown in Table 5, 3% of the 170 non- malarious human sera, specially selected from patients with other disease entities that would give specific positive titres in the IHA test for malaria, had an IHA titre > 1:40 and only about 0.5% of them had an IHA titre > 1: 80 with specifically sensitized cells. This suggests that the IHA test is specific for malaria. It seems unlikely that the sera react with some erythrocyte antigenic determinants freed from the host cells during the process of haemolysis. Otherwise the bovine sera from infections with Anaplasma marginale and the serum from a patient with an idiopathic haemolytic anaemia, after absorp- tion with fresh sheep cells, should both have shown positive titres with the specifically sensitized cells. Moreover, the observations on the malaria infections in rhesus monkeys indicate that the host erythrocyte determinants are not the main part of the sensitizing antigens. These antigens are prepared from para- 780 INDIRECT HAEMAGGLUTINATION TEST FOR MALARIA sitized blood of rhesus monkeys. Nevertheless the monkey serum samples after absorption with cells sensitized with control antigen prepared from nonin- fected rhesus monkey red cell material still showed reactivity with the specifically sensitized cells. The IHA test for malaria seems to be affected more by the influence of heterophile antibodies than by nonspecificity. Adeniyi-Jones (1967) found a high incidence of agglutinins against tanned sheep cell erythrocytes in Nigerian sera. In contrast to the present observation, she reported that agglutination was not affected by absorption with normal sheep cells and that the absorption with tanned cells met with varied success. Kano et al. (1968) found agglu- tinins to guineapig erythrocytes and to rat erythro- cytes in Gambian sera. In both these papers the authors suggest a possible relationship with malaria. This view was opposed by Greenwood (1970) in his study on heterophile antibodies in Nigerian sera. Others have reported on the possible relation be- tween malaria infections and other unusual host res- ponses. Curtain et al. (1965) demonstrated a high incidence of cold agglutination, Houba & Allison (1966) an increased incidence of rheumatoid factor- like antibodies, and Shaper et al. (1968) circulating autoantibodies to heart, thyroid, and gastric parietal cells. In the present study, the relationship in time between malaria parasitaemia and the development of agglutinins against nonsensitized tanned sheep cells was demonstrated in a P. ovale infection and in both blood-induced and sporozoite-induced pri- mate malaria infections in rhesus monkeys. The development of these heterophile antibodies occurred in an unpredictable way. Some patients and animals did not show a rise in heterophile antibody titre. Moreover, some animals produced these antibodies early during the primate parasitaemia while others did so many weeks after the commencement of the patent parasitaemia. The rise in heterophile anti- body titres occurred some time after the increase in the titre of haemagglutinins for specifically sensi- tized cells. About 30% of 276 human sera with an IHA titre >1: 80 for specifically sensitized cells also showed haemagglutinin activity for non-sensitized tanned cells. The significance of the production of these hetero- phile antibodies became apparent after absorption with fresh sheep cells. Some of the sera lost not only their haemagglutinating capacity for non- sensitized cells but also that for specifically sensitized cells. In one serum the absorption was only partly successful. In later experiments, it was shown that certain sera also appeared to have some haemagglutinins that reacted with determinants of the control antigen prepared from rhesus monkey red cell material. Some sera that did not react with nonsensitized tanned sheep cells reacted with cells sensitized with this control antigen. This second type of hetero- phile antibody could in most cases be absorbed with fresh sheep cells but, as is shown in Table 7, not always. Exceptional cases were encountered that had haemagglutinins for tanned cells sensitized with control antigen; these heterophile antibodies could not be absorbed. In view of the possibility that different types of haemagglutinin with different specificities develop during infections with plasmodia, it is necessary to determine to what extent all these haemagglutinins form an antibody family with broad cross-reactivity and with definite affinities, which differ from host to host, for erythrocytes of various species. So far we have not had the opportunity of studying the incidence of heterophile antibodies in the field. Nevertheless it appears from our preliminary studies that both specifically sensitized tanned sheep cells and tanned cells sensitized with control antigen should be used in the IHA test. All sera with haemagglutinins for specifically sensitized cells should be absorbed preferably with cells sensitized with host erythrocytic control antigen and then tested again. Otherwise the interpretation of the IHA titres of malaria sera is likely to be incorrect. In the introduction to this paper a plea was made for a simple serological field test for malaria. The necessity to include a routine absorption procedure in the serological titration involved in the IHA, test detracts from that ideal. It is clear that further studies on the nature of the specific sensitizing anti- gens and on methods for their isolation are necessary. ACKNOWLEDGEMENTS We are indebted to Miss C.rSmeets and Mr J. van Munster for their technical assistance. We thank Pro- fessor P. J. Zuidema, Dr A. Heyen, Dr F. Klein, and Dr G. Stoelinga for the serum samples from different patients and Professor D. Zwart for providing sera from Nigerian cattle with an Anaplasma marginale infection. 781 782 J. H. E. T. MEUW EN ET AL. RPSUMMA ETUDES SUR DIVERS ASPECTS DE L'EPREUVE D'HAMAGGLUTINATION INDIRECTE DANS LE PALUDISME L'6preuve d'h6magglutination indirecte (HAI), appli- qu6e avec tant de succ6s a divers domaines de la micro- biologie et de l'immunologie, en est encore a attendre d'etre admise comme m6thode de routine dans l'6tude s6ro6pid6miologique du paludisme. Depuis quelques ann6es, les auteurs utilisent de temps a autre cette tech- nique au cours de recherches experimentales sur le palu- disme. Cet article constitue un rapport pr6liminaire sur leurs travaux, particuli6rement sur ceux qui visent a perfectionner la m6thode et a appr6cier l'influence qu'exerce sur l'epreuve la formation d'anticorps hMte- rophiles. Les souches et esp6ces de parasites etaient: Plasmodium cynomolgi bastianellii, P. c. langur, P. c. ceylonensis, P. inui, P. knowlesi, P. knowlesi Guy, P. fieldi et P. vinckei. L'antigene sensibilisant a ete obtenu en injectant par voie intraveineuse a des singes rh6sus splenectomises les plasmodiums appropri6s. On procedait a la saign6e au moment oui la densit6 parasitaire etait 'a son maximum, essentiellement sous forme de schizontes. Les cellules sensibilisees etaient des globules rouges de mouton traites 'a 'acide tannique. En pratique, I'antigene sensi- bilisant etait ajoute aux cellules tann6es dans la propor- tion optimale d6terminee par titration de l'antigene. En comparant les r6sultats obtenus par l'HAI et 1'IFI (epreuve d'immunofluorescence indirecte) appliquees aux 276 memes serums, on a constat6 que les deux methodes foumissent un moyen sensible et specifique de suivre la formation des anticorps au cours du paludisme. Les serums avaient et6 choisis arbitrairement parmi des sujets se plaignant de symptomes en relation possible avec cette maladie. Le plus souvent, il n'a pas ete possible de mettre en 6vidence la pr6sence de parasites dans le sang. I1 ressort des r6sultats que, lorsque la quantit6 d'anticorps est faible, les titres obtenus par l'un ou l'autre proc6d6 sont tres voisins. De plus, dans les s6rums humains a reaction positive, les titres d'HAI a l'antigene P. cyno- molgi bastianellii ont tendance a 8tre g6neralement plus forts que ceux trouv6s par IFI avec l'antigene P. fieldi. L'emploi de sept antigenes diff6rents indique que l'HAI d6montre la presence d'anticorps possedant une specifi- cit6 de groupe. Les antigenes P. cynomolgi bastianellii et P. inui se sont rev6l6s les plus sensibilisants dans chaque 6preuve. L'HAI semble etre specifique pour le paludisme. En d'autres termes, c'est plut6t l'intervention d'anticorps heterophiles que le d6faut de specificite qui est genante pour cet examen. Certaines infections comportent egale- ment des agglutinines actives sur les composants des h6maties de l'hote. Comme temoin du titrage des agglu- tinines specifiques par 1'HAI, on preconise l'absorption des serums a l'aide de cellules de mouton tann6es sensi- bilisees par des antigenes d'hematies d'un sujet non infecte. La n6cessite d'adjoindre systematiquement une technique d'absorption au titrage s6rologique par l'HAI complique l'epreuve. II reste bien sfir necessaire de poursuivre les recherches sur la nature des antigenes sensibilisants specifiques et sur les m6thodes propres a les isoler. De ces travaux d6pend le perfectionnement de cette technique s6rologique d'etude du paludisme. REFERENCES Adeniyi-Jones, C. (1967) Lancet, 1, 188 Bray, R. S. (1965) Ann. Soc. belge Mid. trop., 45, 397 Bray, R. S. & El-Naha, H.M.S. (1966) Nature (Lond.), 212, 83 Curtain, C. C. et al. (1965) Brit. J. Haemat., 11, 471. Desowitz, R. S. & Stein, B. (1962) Trans. roy. Soc. trop. Med. Hyg., 56, 257 Greenwood, B. M. (1970) Clin. exp. Immunol., 6, 197 Houba, V. & Allison, A. C. (1966) Lancet, 1, 848 Kano, K. et al. (1968) Proc. Soc. exp. Biol. Med., 129, 849 Mahoney, D. F. et al. (1966) Milit. Med., 131, Suppi. 1141. Meuwissen, J. H. E. Th. (1966) Trop. geogr. Med., 18, 250 Rogers, W. A. et al. (1968) Amer. J. trop. Med. Hyg., 17, 804 Sadun, E. H. et al. (1969) Milit. Med., 134, Suppl. 1294 Sever, J. L. (1962) J. Immunol., 88, 320 Shaper, A. G. (1968) Lancet, 1, 1342 Stavitsky, A. B. (1954) J. Immunol., 72, 360 Stein, B. & Desowitz, R. S. (1964) Bull. Wid Hlth Org., 30, 45 Wellde, B. T. (1969) Milit. Med., 134, Suppl.,1284 Wilson, M. et al. (1971) Amer. J. trop. Med. Hyg., 20, 6 WHO Scientific Group on the Immunology of Malaria Wld Hith Org. techn. Rep. Ser., No. 396