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Keywords:

  • malaria;
  • Plasmodium falciparum;
  • circumsporozoite antibodies;
  • NANP

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Circumsporozoite (CS) antibodies are a reliable serological marker for the infection of Plasmodium falciparum. The purpose of this investigation was to construct and evaluate an enzyme-linked immunosorbent assay test for the detection of CS antibodies. While the sensitivity of the newly developed test reached 78%, the specificity was 99%. In addition, the optimized kit was used to test for infection with P. falciparum in 1903 travellers that were recruited from a prospective study for malaria chemoprophylaxis. Sixty-six of the 1903 patients (3.5%) showed elevated CS antigen antibody titres. However, seroconversion could only be demonstrated in 18 (0.95%) patients. Among those seroconverting, there was a significantly higher percentage of male travellers (1.28%) than female travellers (0.56%). Positive reactions were more frequent among returnees from West and East Africa (1.49 and 1.14%, respectively) than among those from other endemic areas, e.g. South America (n=0). Despite its limited sensitivity, this newly developed kit for CS antibody testing may be a valuable tool for the estimation of the risk for travellers in malarious regions to acquire an infection with P. falciparum. It may also be useful for the determination of the efficacy of malaria chemoprophylaxis for inhibiting outbreak of disease.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Antibodies to the circumsporozite (CS) stage of malaria parasites are a reliable serological marker indicating infection of Plasmodium falciparum even without clinical signs of malaria. Therefore, detection of these antibodies has been used in epidemological studies to estimate the intensity of malaria transmission (Druilhe et al. 1986; Esposito et al. 1988; Hoffman et al. 1986; Del Giudice et al. 1987). Antibodies bind to the immunodominant epitope of the CS-surface protein. This CS protein consists of highly conserved tandem repeats of amino acids (NANP=Asn-Ala-Asn-Pro) (Zavala et al. 1986). Peptides of different length were used in enzyme-linked immunosorbent assay (ELISA) tests and commercial ELISA-kits were used (e.g. NANP50, NANP40, NANP4) (Zavala et al. 1986; Esposito et al. 1990).

In individuals living in endemic areas, prevalence and levels of sporozoite antibodies have been shown to correlate with the entomological inoculation rate assessed at the same time for the same area (Esposito et al. 1988). Because of significant differences in living conditions and use of protective measures in travellers to malarious regions, the risk of infection with P. falciparum may vary considerably from that of the local population. Detection of CS antibody titres lends itself as rapid surrogate marker for measurement of the infection rate among travellers protected by chemoprophylaxis. In order to estimate the actual infection rate for travellers in malarious regions, this study examined the prevalence of CS antibodies in a large number (1903) of travellers that were recruited for a chemoprophylaxis study in 1999. For this purpose, a new test kit using NANP19 as antigen was constructed and evaluated.

Patients, materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Test kit

The NANP19 antigen (synthesized by Hoffmann la Roche, Switzerland) (Döbeli et al. 1998) was diluted in phosphate-buffered saline (PBS, pH 7.2) from which 100 μl (5 μg/ml) were applied per well of a 96-well microtitre plate (Dynatech Immunolon II). The plate was coated for 1 h at 37 °C following an overnight period at 4 °C. After washing with PBS + 0.1% Tween the wells were blocked for 1 h at 37 °C with 200 μl PBS–5%-MP (PBS + 5% milk powder) and were then washed again. Sera were diluted 1 : 250 in PBS–0.5%-MP and applied 100 μl/well for 1 h at 37 °C and washed afterwards. The conjugate [antihuman IgG (h + l) goat] was diluted 1 : 16000 in PBS–0.5%-MP and applied 100 μl/well for 1 h at 37 °C following washing and rinsing of the wells with Aqua dest. Then, 100 μl of the substrate buffer was added (one tablet OPD: orthophenylenediamide dissolved in 50 ml NaH2PO4/Na2HPO4 buffer at pH 5.0). After 10 min the enzymatic reaction was stopped by adding 25 μl 8 M H2SO4. Absorbance was read at 492 nm.

Evaluation

Four different panels of sera from patients presented to the outpatient clinic of the Department for Infectious Diseases and Tropical Medicine in Munich were investigated. The first group (n=118) consisted of patients with P. falciparum malaria. Diagnosis was confirmed microscopically by thin and thick blood-films in all patients. The sera were obtained between 1 and 90 days after onset of symptoms. The second group (n=68) was formed by European patients with different tropical diseases (malaria tertiana n=22; amoebiasis n=10; schistosomiasis n=10; dengue n=10; leishmaniasis n=7; filariasis n=6; Chagas disease n=3). The third group (n=117) consisted of persons who had never been to malarious areas previously, the fourth (n=50) consisted of people with positive rheumatoid factor, who had never been to endemic areas, either.

Prospective study

Sera of 1903 persons were taken before and after their travel to malaria endemic countries during a large study for malaria chemoprophylaxis. Study conditions and recruitment have been described elsewhere (Overbosch et al. 2001). The study group consisted of 891 female (47%) and 1012 male (53%) travellers. The age ranged from 5 to 80 years, the average was 34.5 years (median: 31 years). Duration of travel extended from 2 to 38 days with an average of 18 days (median: 17). Destinations were East Africa n=614 (32%), West Africa n=402 (21%), Southern Africa n=272 (14%), South America n=149 (8%), Central Africa n=56 (3%), and other malarious areas n=410 (22%). Data were stored in a database (MS Excel®) and analysed by Epi Info (WHO & Geneva 2000) for statistical significance.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Evaluation

The first group tested in this study consisted of 118 patients with falciparum malaria. Positive reactions could be observed in 80 specimens (68%). In the first 10 days after onset of symptoms, 31 of 55 specimens were positive (56%) while during days 11 and 90 after onset of symptoms, 49 of 63 specimens were positive (78%). The cut-off value for measurements of CS antibodies was defined as 3 units [U]. The mean value of the antibodies concentration in positive specimens during the first 10 days was 11.4 U and between days 11 and 90 it was 24 U (Table 1). The second group (n=68) consisted of patients with a variety of parasitic diseases. Positive reactions could be observed in two out of 22 specimen with vivax malaria (9%) and in two out of 10 specimen with filariasis (20%). There was no positive reaction in patients with amoebiasis, dengue, Chagas disease, leishmaniasis and schistosomiasis. Of all the patients who had never been to malaria endemic areas, there was one of 117 patients who reacted positive (0.85%) in the CS-ELISA (mean: 0.86 U). All 50 patients with rheumatoid factors reacted negative (mean: 0.95 U).

Table 1.  Evaluation of a NANP19 test for detection of Plasmodium falciparum anticircumsporozoite antibodies with a batch of serum samples from malaria patients Thumbnail image of

Prospective study

In the group of 1903 travellers that were investigated for the prevalence of CS antibodies, a total number of 66 patients (3.5%) had significant antibody titres when tested after return from their journey (P < 0.0001; Mantel Haenszel test). However, a seroconversion could only be demonstrated in 18 of 1903 specimens (0.95%). The mean value of antibody titres in the group of the seroconverters was 1.6 U before and 5.9 U after travel. Forty-eight of the 1903 participants (2.5%) already showed a positive reaction in the CS ELISA before their journey. The age average in the subgroup of seropositive travellers was 31 years (median: 25 years, range: 8–72), 72% were male (P=0.0056; Mantel Haenszel test). The duration of travel was 5–32 days, 17 days on average (median: 16 days) (Table 2). Travel destinations are shown in Table 3. All seroconverters travelled to highly endemic regions: East Africa n=7 (39%), West Africa n=6 (33%), Southern Africa n=2 (11%), and other countries n=3 (17%). None of the travellers to Central Africa and South America seroconverted.

Table 2.  Testing for anti-CS antibodies in travellers: comparison of seroconverters with the total study population Thumbnail image of
Table 3.  Testing for anti-CS antibodies in travellers: travel destinations in seroconverters and the total study population Thumbnail image of

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Circumsporozite antibodies, indicating the mere fact of infection, have been shown to be reliable indicators of transmission in endemic areas (Druilhe et al. 1986; Hoffmann et al. 1986; Del Giudice et al. 1987; Esposito et al. 1988; Archibald et al. 1990; Webster et al. 1992; Ferreira et al. 1995; Jelinek et al. 1995; Camargo et al. 1996; Bwire et al. 1998; Cobelens et al. 1998). Though prevailing ELISA tests have shown low sensitivity, the method can be a reliable test for estimating the risk of infection for travellers (Jelinek et al. 1995). These findings are reflected in the newly developed NANP19 kit that was evaluated in this study. Depending on the time of blood taking, the sensitivity ranged from 56% (during the first 10 days after onset of symptoms) to 78% (days 11–90). Thus, for optimized use of the kit in asymptomatic returnees from endemic areas, testing of blood samples should be recommended for the time window of 10–90 days after return from the journey. Only one of 117 specimens of the malaria-negative control group reacted positive for CS antibodies. Although patients with positive rheumatoid factors usually have a high potential for cross-reactions in serological testing, no positive reactions occurred in this control. A limited number of cross-reactions were observed in patients positive for Plasmodium vivax antigen (9%) and with filariasis (20%). In weighing these results, it should be considered that an additional P. falciparum infection can be acquired easily in most regions endemic for the above-named parasites. Similar results for serological cross-reactions with P. vivax antigen have been observed elsewhere with related test kits (Jelinek et al. 1995). In view of the evaluation of this test kit, its satisfying sensitivity and specificity for the detection of an infection with P. falciparum can be assumed. Previous test kits reached a sensitivity of 55.8% and a specificity of 100% (Jelinek et al. 1995).

In the group of 1903 travellers to malarious areas, the percentage of specimens with a seroconversion for CS antibodies (0.95%) correlates well with previous estimates of the overall infection rate for travellers with P. falciparum in endemic regions (Mühlberger et al. 1998). Regions where travellers are considered to be at high risk were reflected in the geographical distribution of infection rates: while West Africa (1.49%) and East Africa (1.14%) contributed a higher proportion of seroconverters than the total rate (0.95%), a lower percentage of travellers was infected in Southern Africa (0.74%), South America (0%) and other areas (0.73%). This corresponds well with reports from surveillance systems on imported falciparum malaria to Europe (Jelinek et al. 2002). The higher proportion of male travellers who were infected with P. falciparum (1.28 vs. 0.56% female travellers) may be explained by different behaviour and thus increased exposure to the vector. Gender-related differences in the length of travel were not observed. Similar proportions have been described elsewhere (Kriechbaum & Baker 1996; Mühlberger et al. 1998). The relatively high number of persons (n=48; 2.5%) who provided positive serum samples prior to their journey can be explained by the recruitment procedure for the prophylaxis study: previous travel to malarious areas was not an exclusion criterion, and they may have harboured remaining CS antibodies from former journeys. Positive CS antibody titres drawn 880 days after onset of malaria symptoms were described previously (Jelinek et al. 1996). However, this particular result also emphasizes the importance of having serum samples for determination of a pre-travel baseline before testing for CS antibodies as a tool for the measurement of the malaria pressure on travellers in a given area. The time of exposure in malarious areas is strongly linked to CS antibody response (Druilhe et al. 1986; Webster et al. 1992; Jelinek et al. 1996). This fact may be the reason for a lower percentage of seroconverters in this investigation compared with earlier studies. The duration of travel in our group of 1903 travellers was comparatively short with an average of 18 days (range 2–38 days).

This study has demonstrated that testing for CS antibodies with the help of an NANP19 ELISA test can be a reliable marker for the prospective detection of an infection with P. falciparum, regardless of malaria being clinically manifest or not. The method can also help determining risks for acquiring P. falciparum infection in different geographical areas. An estimate for the need of malaria chemopropylaxis in certain areas can be provided. In addition, this test can be a valuable tool for controlling the efficacy of chemoprophylaxis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported in part by the Boehringer Ingelheim Fonds. We explicitly thank Heinz Döbeli, Hoffmann La Roche in Basel, Switzerland, for donating the NANP19 for this research and Hans Peter Beck, Swiss Tropical Institute, Basel, Switzerland, for the very fruitful collaboration at early stages of this project. Glaxo Smith Kline in London, UK, provided the serum samples for the prospective part of the study.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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