Determination of malarial antibodies by means of screening and differentiating antigens

ZbI. Bah. Hyg., I. Abt, Orig. A 252, 566-571 (1982) Department of Bacteriology and Serology, Bernhard-Nochr-Institure for Maritime and Tropical Diseases, Hamburg

Determination of Malarial Antibodies by Means of Screening and Differentiating Antigens Nachweis von Malaria-Antikorpern mit Hilfe von Such- und differenzierenden Antigenen

JORGEN KNOBLOCH, ERICH MANNWEILER, and INGEBORG ZUM FELDE Received March 3, 1982

Abstract 4455 human sera with malarial antibodies were analysed with regard to their reactivity screening antigens (Plasmodium fieldi and P. [alciparum) and to differentiating antigens (P. uiuax, P. ovale and P. malariae lbrasilianum ). Results were obtained by indirect fluorescent antibody test. The identification rate of antibodies was significantly higher with P. fieldi than with P. [alciparum antigen, and was lowest for both of the screening antigens in sera with antibodies to P. ovale. The highest cro ss reactivity wa s between P. vivax and P. fieldi, the lowest between P. [alciparum and P. fieldi. The majority of sera not reactive to screening antigens was reactive to P. ovale. The reactivity of sera to one of the screening antigens was higher in the group of immigrants from malaria endemic areas in comparison to European travellers, the overall pattern of results, however, was similar in both groups of patients. to

Zusammenfassung 4455 menschliche Seren mit Malariaantikorpern wurden hinsichtlich ihrer Reaktionsbereitschaft mit Suchantigenen (Plasmodium fieldi und P. [alciparum) und mit differenzierenden Antigenen (P. uiuax, P. ovale und P. malariaelbrasilianum] mit Hilfe der indirekten Immunfluoreszenz untersuchr, Mit P. fieldi wurden signifikant haufiger Antikorper nachgewiesen als mit P. [alciparum als Antigen. Die Reakrivitar der Suchantigene war am geringsten in Seren mit P. ovale-Antikorpern. Die srarksten Kreuzreaktionen traten zwischen P. vivax und P. fieldi auf, die geringsten zwischen P. [alciparum und P. fieldi. Die Mehnahl der Seren, die nicht mit den Suchantigenen reagierten, hatren Antikorper gegen P. ouale. Bei Einwanderern aus Gebieten mit endemischer Malaria waren im Vergle ich zu europaischen Tropenreisenden signifikant haufiger Anrikorper mit einem der beiden Suchantigene nachzuweisen; das Verteilungsmuster der Anrikorpcrbildung war jedoch in beiden Gruppen ahnlich,

Determination of Malarial Antibodies

567

Introduction

The indirect immunofluorescence antibody test is considered to be the reference method for malarial immunodiagnosis and seroepidemiology (21). As homologous antigen is not always available, simian plasmodia have been used successfully for the detection of human antibodies to malaria (2, 6, 8, 10, 12, 14, 15, 18, 20). Cross reaction occurring to varying degrees between different plasmodium species have been used to establish an economical combination of antigens for the screening of human sera for malarial antibodies (3,5, 7, 9, 13, 16, 19). In this paper we report the reactivity of 4455 seta to malarial antigen, utilizing Plasmodium fieldi and P. [alciparum as screening and P. ovale, P. uiuax, P. malariae and P. brasilianum as differentiating antigens.

Material and Methods Sera: The specimens were sent to our laboratory by different German hospitals and private physicians. The respective patients were either immigrants from malaria endemic areas or Europeanstemporarily exposedto malaria. Allsera containing malarial antibodies, as tested over a 4 years period, were included in the study. Antigen: P. fieldi and P. brasilianum derivedfrom monkeys P. ovale, P. vivax, P. malariae and P. falciparum were collectedfrom patients presenting the rexpective parasitaemia. Serological testing: Indirect immunofluorescence antibody test (IFA) was performed accordingto Sulzer et al. (1969). The serum-dilutions studied ranged from 1 :20 to 1: 1280. Statistical calculation was performed by chi-square test.

Results All sera were screened by P. [alciparum and P. fieldi antigen. Other than these antigens were used for screening, when the attending physician asked for it because of suggestive symptoms or known history. Thus 4455 sera with malarial antibodies were analysed, of which 41 % had antibodies to P. fieldi only, 22% to P. falciparum only and 36% to both P. fieldi and P. falciparum. The sera which were not reactive to the screening antigens (14) but reactive to differentiating antigens are listed in Table 2. The percentage of sera reactive to both of the screening antigens was significantly higher (p < 0.01) in the group of patients originating from malaria-endemic areas as compared to temporarily exposed Europeans (Table 1). By far most of the specimens neither reactive to P. fieldi nor to P. [alciparum antigen reacted with P. ovale only. However, single sera reacted with up to three differentiating antigens but not with screening antigens (Table 2), presenting titres that ranged from 1: 20 to 1: 1280. In order to determine the efficiency of the screening antigens, results obtained by differentiating antigens were compared to those obtained by using P. fieldi and P. [alciparum antigen for the same sera. P. malariae and P. brasilianum were comprised within the same group because of their antigenic relationship (12, 17). Table 3 gives the respective results in comparison of temporarily exposed Europeans to immigrants from malaria endemic areas. Significantly more malaria antibody con-

j.Knobloch, E.Mannweiler, and 1. zum Felde

568

Table 1. Human sera with malaria antibodies* obtained by indirect immuno-fluorescence antibody test Group

Percentages of 4455 human sera with antibodies against antigens P. [alciparum P. [alciparum other antigens,'* only only + P. fieldi P. fieldi

Temporarily exposed Europeans Residents of endemic areas Total

47

22

30

1

21

23

56

0

41

22

36

1

" Titre range: 1: 20 to 1: 1280 ,',' P. ovale, P. uiuax, P. malariae (see Table 2)

Tab.. ') sera non-reactive p asrnodium antigens

n

to

P. fieldi or P. [alciparum antigen, but reactive to other

P.ovale

only

P. vivax only

P. malariae P.ovale P. ovale only + P. vivax + P. mal.

P. vivax + P.mal.

P.ovale + P. vivax + P. mal.

32

2

2

1

1

5

2

taining sera were detected by using P. fieldi antigen than by P. [alciparum antigen in either group of patients. The high rate of P. [alciparum antibody containing sera which were not or less reactive to P. fieldi antigen (63% and 53%, respectively) and vice versa (65% and 37%, respectively) indicates the considerable antigenic difference between these screening antigens. Cross reactivity was more distinct between the differentiating antigens (P. uiuax, P. ovale and P. malariae/brasilianum) and the screening antigens (P. fieldi and P. falciparum). However, four to fifty percent of sera reactive to differentiating antigens did not react with one of the screening antigens (Table 3). The antigenic relationship between P. vivax and P. fieldi was indicated by the fact that 90 to 96% of the sera with P. vivax antibodies were reactive to P. fieldi antigen. Least efficient, on the other hand, was P. [alciparum antigen for the detection of malarial antibodies in sera reactive to P. ouale in the group of European travellers (Table 3). With the exception of the sera with P.ovale antibodies reacting with P. fieldi antigen, the percentage of sera reactive to screening antigens was always significantly higher in residents of tropical areas when compared to temporarily exposed Europeans.

Immigrants from endemic areas

Temporarily exposed Europeans

773

799 157 262 143

2653

P. P. P. P. P. P.

[alciparum vivax ouale malariae brasilianum fieldi

-

29 4 9 6

11 11

24 14 26 32

21 19 32 16

47 82 65 62

57 73

71

37

42 10

1775 310 625 141

P. [alciparum P. vivax P. ovale P. malariae P. brasilianum P. fieldi

Sera reactive to P. fieldi antigen (%) negative less reacti ve equally or more reactive

Sera reactive to differentiating antigens n Antigen

27

11

17 20

61

43 50 28

10

15 10 19

4

12 9 11

63

68 70 70

35

45 41 61

Sera reactive to P. [alciparum antigen (%) negative less reactive equalIyor more reactive

Table 3. IFA-antibodies against antigens of screening and differentiating plasmodia in sera from exposed Europeans and immigrants from endemic areas

'"

0\

v,

o»

0-

o r;"

&'

E >g

$I>

...Pi"

~

a.

~

o'

~

S'

t:I

"rti 8

570

j.Knobloch, E.Mannweiler, and 1. zum Felde

Discussion Serological testing in malaria is useful for seroepidemiological studies, for the screening of blood donors, for case-detection when parasitemia is low and for retrospective diagnosis (6, 7, 14). In any case information is necessary about the diagnostic efficiency of screening antigens including their relations to differentiating antigens. In this regard we analysed sera of patients who had developed antibodies due to naturally acquired malaria infection, utilizing the simian P. fieldi and P. [alciparum as screening antigens. Sensitivity and specifity of our IFA test system was previously evaluated by comparison with control groups (8, 10). Thus a titre of 1: 20 or more indicating specific antibodies was demonstrated. We emphasize that malarial antibodies develop as a result of parasitaemia, however, not necessarily indicate previous clinical malaria. As expected, there was a more frequent reactivity to screening antigens in sera of immigrants from tropical areas (Table 1 and 3), which reflects a higher frequency of previous infections as compared to European patients (4,6). Cross reaction occurring between different plasmodial antigens have been previously observed (2, 5, 6, 8, 10, 12, 13, 14, 15, 18, 20). Thus the serologist was enabled to apply even simian antigens to the serology of malaria, such as P. fieldi and P. brasilianum. An efficient and economic combination of screening antigens is desirable. Our data suggest that P. fieldi and P. [alciparum in this regard serve as good supplements, since the cross reactivity was lower than between the differentiating and the screening antigens (Table 3). However, in some of our sera malarial antibodies could be identified only by differentiating antigens (Table 2), which is apparently explained by antigenic differences among strains, occasionally occurring as an intra strain variation in a single chronic infection (1, 9). In our cases it was mainly P. ovale antibody that did not react to screening antigens. Significantly more sera with plasmodial antibodies were identified by P. fieldi antigen than by P. [alciparum, Particularly there was an intense cross reactivity between P.vivax and P.{ieldi which was previously reported by other authors (6, 8, 10, 15). Nearly as reliable seemed to be the identification of antibodies to malaria quartana by P. fieldi antigen. The least identification rate was achieved by both P. fieldi and P. [alciparum antigen in sera with antibodies to P. ovale. It is noticeable that the screening antigens were more efficient in the group of immigrants from malaria endemic areas than in the group of European travellers concerning the detection of malarial antibodies, but that the distribution of reactivity rates was similar in both groups of patients (Table 3). We conclude that P. fieldi and P. [alciparum antigen represent a recommendable combination for the screening of human sera with plasmodial antibodies. It has to be considered, however, that false negative results do occur when using these screening antigens, particularly in cases of P. ovale infection. References 1. Brown, K. N.: Antigenic variation and immunity to malaria. In: Parasites in the immun-

ized host: mechanism of survival. Ciba Symposium Nr. 25 (1974) 35-51 2. Collins, W. E., G. M. Jeffery, E. Guinn, and]. C. Skinner: Fluorescent antibody studies

Determination of Malarial Antibodies

571

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