75 Am. J. Trop. Med. Hyg., 58(1), 1998, pp. 75–80 Copyright  1998 by The American Society of Tropical Medicine and Hygiene TESTING FOR ANTI-CIRCUMSPOROZOITE AND ANTI–BLOOD-STAGE ANTIBODIES FOR EPIDEMIOLOGIC ASSESSMENT OF PLASMODIUM FALCIPARUM INFECTION IN TRAVELERS FRANK G. J. COBELENS, JAN-PETER VERHAVE, ANNE LEENTVAAR-KUIJPERS, AND PIET A. KAGER Division of Infectious Diseases, Tropical Medicine and AIDS, Department of Internal Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Department of Medical Parasitology, University of Nijmegen, Nijmegen, The Netherlands; Section of Public Health and Environment, Municipal Health Service, Amsterdam, The Netherlands Abstract. The purpose of this investigation was to assess the role of serology for establishing incidences of Plasmodium falciparum malaria and of exposure to P. falciparum in epidemiologic studies of travelers using che- moprophylaxis. The design was a prospective cohort study involving 548 short-term Dutch travelers to areas endemic for P. falciparum malaria. Sera were collected before departure and, together with the medical history, 2–6 weeks after return. All sera were tested for anti-circumsporozoite (CS) antibodies by an R32tet 32 -ELISA; sera of subjects reporting febrile illness during travel or after return or with anti-CS responses were tested for anti–blood-stage antibodies by an indirect fluorescence antibody test (IFAT). Five subjects (0.9%) reported P. falciparum malaria confirmed by thick blood smear examination (documented cases) and six (1.0%) reported treatment for malaria without a documented diagnosis (presumptive cases). Conversions in the IFAT were detected in six subjects, including all five documented cases and one presumptive case. Anti-CS antibodies were detected in seven subjects (1.3%), including three documented cases and four of 442 subjects with no history of fever or malaria treatment (0.9%). Incidence rates per 1,000 person-months of travel (95% confidence interval) of infection with P. falciparum, whether or not suppressed by chemoprophylaxis, were 16.9 (8–31) for all destinations and 91.6 (33–200) for West Africa. In epi- demiologic studies of P. falciparum malaria in travelers, testing for antibodies to blood stages can increase the sensitivity and specificity of case detection; testing for antibodies to sporozoites may be useful for the assessment of exposure to P. falciparum in travelers using chemoprophylaxis, but the sensitivity is limited. In most epidemiologic studies that assess risks of Plas- modium falciparum malaria or efficacies of prophylactic measures in nonimmune travelers, incidences are based ret- rospectively on cases of malaria that are confirmed by blood slide evaluation. 1–5 This method is likely to underestimate true incidences of malaria because antimalarial treatment is frequently started without reliable parasitologic confirma- tion. In addition, this method will result in underestimation of the exposure to P. falciparum when the travelers studied take chemoprophylaxis. Since chemoprophylaxis suppresses the development of blood-stage parasites, infection by spo- rozoites will generally not progress to patent infection and clinical disease. Therefore, data based on blood slide results tend to reflect risks of breakthrough malaria or of malaria in individuals taking inadequate prophylaxis, and are con- founded by drug resistance patterns and compliance with prophylactic regimens. Serologic methods may have value in studies aimed at establishing malaria risks or efficacy of prophylactic mea- sures in travelers by identifying individuals with recent ex- posure to sporozoites and/or by retrospective confirmation of diagnoses of malaria. Tests for antibodies to blood stages of P. falciparum are frequently used in seroepidemiologic studies of endemic populations. 5–7 One report has illustrated the application of the indirect fluorescence antibody test (IFAT) for retrospective diagnosis of malaria in travelers who took stand-by antimalarial treatment. 8 Antibody re- sponses to sporozoites have been studied extensively in im- mune populations 9–11 and in nonimmune individuals, 12, 13 mostly by ELISAs based on synthetic or recombinant pep- tides from the repetitive domain of the circumsporozoite (CS) protein. Recently, a retrospective study reported a high prevalence (21%) of anti-CS antibodies in asymptomatic travelers returning from sub-Saharan Africa. 14 We studied if testing for antibodies against the CS protein by R32tet 32 -ELISA and against blood stages by IFAT would improve the assessment of exposure to P. falciparum and the detection of cases of P. falciparum malaria in a cohort of travelers to endemic areas who were taking chemopro- phylaxis. SUBJECTS AND METHODS The study was conducted at the travel immunization clinic of the Amsterdam Municipal Health Service. Between Feb- ruary 1991 and February 1992, all Dutch-speaking clients 15 years of age and older who intended to travel to tropical or subtropical destinations for 1–13 weeks were asked to participate. The analyses reported here included only sub- jects who traveled to areas endemic for P. falciparum ma- laria. The study was approved by the Service’s Board of Medical Ethics. Before departure, a blood sample was taken and a questionnaire that addressed travel history and plans was completed. All subjects were prescribed proguanil, 100 mg/day, and chloroquine, 300 mg/week, for chemoprophy- laxis. The daily proguanil dose was doubled for all subjects enrolled after July 1991 due to a change in the chemopro- phylaxis guidelines in The Netherlands. Subjects visited the clinic again within six weeks after return from the endemic area for a second blood sample and questionnaire. The post- travel questionnaire addressed travel itinerary, febrile ill- nesses, medical consultation, diagnosis, treatment, and use of chemoprophylaxis. Eligible travelers were included in the study only after having given written informed consent based on a information sheet on the study covering objec- tives, procedure, laboratory investigations, and confidenti- ality. Data ascertainment and definitions. Travel destination