Application of the indirect haemagglutination test for malaria: reproducibility with Plasmodium falciparum-sensitized cells

Bull. Org. mond. Sante' 1973, 49, 317-319 Bull. Wld Hlth Org. Application of the indirect haemagglutination test for malaria : reproducibility with Plasmodium falciparum-sensitized cells J. H. E. T. MEUWISSEN,1 Anna D. E. M. LEEUWENBERG,1 W. N. DOESBURG,2 & J. A. M. VAN DRUTEN2 Abstract The reproducibility of the IHA test was assessed. Two batches of glutaraldehyde-fixed sheep cells were used and the cells were sensitized with different batches ofP. falciparum antigen obtainedfrom 4 owl monkeys. At the individual level the IHA test appears to have a reproducibility of approximately 90 %. Data on the reproducibility of the indirect haemag- glutination (IHA) test for malaria are required for the assessment of the reliability of the test as such and for the evaluation of longitudinal sero-epi- demiological surveys. We report here the test results obtained with a standard set of sera examined with test cells sensitized with P. falciparum antigens obtained from 4 different owl monkeys (Aotus trivirgatus s.l.). Materials and methods For several years we have stored at -20'C serum samples that were received for examination with the indirect fluorescent antibody (IFA) test. These sam- ples are from people who had visited malarious areas and who had a history of malaria parasit- aemia, fever of unknown origin, splenomegaly, gen- eral malaise, or other symptoms that could be related to malaria. For the preparation of a sero- positive test battery, the 73 largest samples were selected that had reacted with titres >1: 40 in the IFA test with P. fieldi as antigen. Four different batches of antigen were used for. the preparation of test cells. P. falciparum antigen was prepared in the usual way from 4 different blood-inoculated owl monkeys, of which Mon- key Ao2 was infected with the West African strain 1 Laboratory of Medical Parasitology, University of Nijmegen, The Netherlands. ' Statistical Department, University of Nijmegen, The Netherlands. and monkeys Ao6, Ao8, and Ao9 with the Palo Alto (East African) strain. The IHA test was performed according to the standard procedure as described earlier in this series, including absorption with control cells sensitized with control antigen. The control antigen was pre- pared from a pool of erythrocytes collected from monkeys Ao6, Ao7, and Ao9 before their experi- mental inoculation with plasmodia. Sheep red blood cells fixed with 1,5-pentanedial (" glutaraldehyde ") (sheep 22) were used as carrier cells. One batch of red cells (collected 7 June 1972) was used in the preparation of test cells with Ao6 and Ao9 antigen and was applied on the same day. This batch of red cells was also used in the prepara- tion of test cells with Ao2 and Ao8 antigen. Another batch of red cells (collected 11 April 1973) was used in a duplicate test with Ao8-sensitized test cells. The latter batch is indicated as Ao8' test cells. IHA titres <1: 40 are considered as negative. The mean titre index is the arithmetic mean of the indi- vidual titre indices. The latter are obtained by divid- ing the reciprocal titres by 10 and calculating the exponent to the basis of 2. Titres <1: 40 were given the index 1, titres of >1: 5120 the index 10. Results Table 1 shows the titre scores obtained with the 5 different batches of test cells. The titre distribution is well spread in all 5 tests. Negative results occurred most frequently with Ao6 test cells (26%). With the others they varied between 11 and 15 %.. The mean titre index varies accordingly from 4.4 with Ao6 test cells to 5.1 with Ao9 test cells. In Table 2 a mutual comparison is given of the 4 examinations in which the same batch of fixed sheep cells was used, sensi- tized with 4 different batches of P. falciparum antigen. In the various comparisons, 84-96% of all observations were within a range of ±11 titre. All tests negative with the Ao9 batch were also negative with the Ao2, Ao6, and Ao8 batches. Only 3119 - 317 BRIEF COMMUNICATIONS Table 1. Titre distribution in a standard set of 73 sera examined by IHA for malaria with 4 different batches of P. falciparum (Aotus) antigen Test cells Titre Ao2 Ao6 Ao8 Ao8' Ao9 < 1:40 11 19 10 11 8 1:40 8 6 8 6 8 1:80 7 6 7 12 9 1:160 11 6 8 7 6 1:320 6 11 11 8 6 1:640 10 8 8 11 15 1:1280 9 4 8 5 6 1 :2560 3 5 4 2 4 1:5120 5 4 4 5 5 >1 :5120 3 4 5 6 6 mean titre index 4.7 4.4 4.9 4.8 5.1 standard deviation 2.7 2.9 2.7 2.8 2.8 one of these negatives was found positive (titre 1: 40) with the Ao81 batch. The results of the examinations with cells sen- sitized with P. falciparum West African strain (Ao2) compare very well with those obtained with cells sensitized with the Palo Alto strain. In Table 3 a comparison is made of the results obtained with 2 batches of fixed sheep cells sen- sitized with the same batch of antigen (Ao8). In two-thirds the titres were identical; only 2 sera (3 %) showed a difference of 2 titres. Finally, we compared the IHA titres obtained with Ao8 test cells with those obtained with the other 4 batches of test cells (Table 4), in order to assess the variability of individual titres. At the indi- vidual titre levels observed with Ao8 test cells, 89-100% (average 93%) of the duplicate tests with the other batches of test cells gave titres that were identical or differed by only one step. Discussion Crude antigen, obtained by the lysis of monkey erythrocytes infected with P. falciparum, is used for the preparation of test cells in the IHA test for malaria. The antigenic constituents of this crude antigen have not yet been purified and defined. It is not yet possible to use exactly measured quantities Table 2. Mutual comparisons of IHA titres of 73 sera examined with the same batch of fixed sheep cells, sensitized with 4 different batches of P. falciparum (Aotus) antigen Difference Mutual comparison in titre Ao9-Ao2 Ao9-Ao6 Ao9-Ao8 nil 38 28 38 + 1 titre 20 85% 31 84% 24 96% - 1 titre 4 2 8 > 2titres 9 12 2 62titres 2 0 1 Ao8-Ao2 Ao8-Ao6 nil 44 33 + 1 titre 17 93 % 28 92 % - 1 titre 7 6 >2 titres 3 6 S 2 titres 2 0 Ao6-Ao2 nil 31 + 1 titre 11 86% - 1 titre 21 > 2 titres 2 < 2 titres 8 of antigen for the preparation of test cells. An opti- mum sensitization is sought with the antigen titration test. It was therefore necessary to assess to what extent the use of antigen batches obtained from different monkeys influenced the variability of the Table 3. Comparison of IHA titres of 73 sera examined with 2 batches of fixed sheep cells, sensitized with the same batch of P. falciparum (Aotus) antigen Difference in titre Mutual comparison ofA08-Ao8 l nil 48 + I titre 8 97 % - 1 titre 15 + 2 titres 1 - 2 titres 1 318 BRIEF COMMUNICATIONS Table 4. Results of IHA examination of 73 sera with P. falciparum-sensitized test cells of Ao8, compared with results obtained with Ao2, Ao6, Ao8 ', and Ao9 test cells. No. Differences in titre Tte of reactions________________________________with Ao8 testacells -3 - 2 -1 0 + 1 + 2 > + 3 % ± 1 titre < 1:40 10 0 0 0 36 3 1 0 97 1:40 8 0 0 9 15 7 1 0 97 1:80 7 0 3 8 15 2 0 0 89 1:160 8 0 2 9 17 3 1 0 91 1:320 11 1 2 9 19 12 1 0 91 1:640 8 0 0 9 18 5 0 0 100 1:1280 8 0 1 12 12 6 1 0 94 1 :2560 4 0 1 6 6 3 0 0 94 1 :5120 4 0 1 1 11 3 0 0 94 >1:5120 5 0 0 2 18 0 0 0 100 Total 73 1 10 65 167 44 5 0 93 IHA test. We also assessed the influence of the use of different batches of sheep cells fixed with 1,5-pen- tanedial on the variability of the test. The results indicate a fair reproducibility. The differences in mean titre levels of the test battery, examined with the various test cells, were small. The reproducibility of individual titres averages about 90% when a range of ± 1 titre step is used as a measure of reproducibility. This study provides a basis for the assessment of other batches of antigen and test cells. Moreover, these data are needed for the evaluation of sero-epidemiological surveys. 319