Prevention of malaria.

Malaria is one of the most prevalent infectious diseases worldwide; it is transmitted in over 100 countries, and it is a major cause of serious morbidity and mortality in travelers. Clinicians should inform travelers of their risk, teach them the principles of personal protection, and offer individualized chemoprophylaxis regimens. The increasing prevalence of multiple drug-resistant Plasmodium falciparum in many parts of the world makes nonpharmacologic methods of malaria prevention important.

The susceptibility of the Indonesian I/CDC strain of Plasmodium vivax to chloroquine.

A strain of Plasmodium vivax from Indonesia was adapted to splenectomized Aotus and Saimiri monkeys and tested for its susceptibility to chloroquine. Animals were infected by intravenous inoculation of heparinized parasitized blood and subsequently treated with 8 or 15 mg (base) of chloroquine by oral intubation. Recrudescence of infection occurred in 4 of 4 Aotus and 5 of 6 Saimiri monkeys treated with 15 mg base of chloroquine, indicating a level of resistance between that of the standard Chesson strain of P. vivax and the recently reported resistant strains from Papua New Guinea.

Safety and immunogenicity of a Plasmodium falciparum sporozoite vaccine: boosting of antibody response in a population with prior natural exposure to malaria.

Recombinant sporozoite vaccine or placebo were administered once to 25 volunteers from an area endemic for malaria. Antibody to R32tet32 rose in 9 of 15 receiving vaccine and remained elevated in 6 for 6 months. Mean absorbance increase was 0.43 +/- 0.40 with vaccine, 0.01 +/- 0.23 with placebo, and 0.72 +/- 0.19 in responders. Six non-responders had significantly lower pre-immunization levels (0.07 +/- 0.05) than responders (0.39 +/- 0.25). There was an association between an increase in immunofluorescence (n = 4) and an increase in absorbance (n = 9) among vaccine recipients (n = 15). Vaccine-induced increase in antibody to natural circumsporozoite antigen was indicated by increases in immunofluorescence and by increases in circumsporozoite precipitation score in 2 of the 5 responders with highest antibody increase measured by enzyme-linked immunosorbent assay. Response to subunit sporozoite vaccine paralleled response to prior natural sporozoite exposure and was significant and prolonged in a population with prior natural exposure to malaria.

Antibodies to Plasmodium falciparum ring-infected erythrocyte surface antigen and P. falciparum and P. malariae circumsporozoite proteins: seasonal prevalence in Kenyan villages.

Two cross-sectional surveys of 954 persons in Asembo Bay and Got Nyabondo, western Kenya, were performed in August-September 1986, after long rains, and in February-March 1987, after a comparatively dry season. Serologic testing was performed using an ELISA with synthetic peptides representing repeat amino acid sequences of the Plasmodium falciparum ring-infected erythrocyte surface antigen (RESA), (EENV)5, (EENVEHDA)4, and (DDEHVEEPTVA)2 and repeat sequences (PNAN)5 and (NAAG)5 of the P. falciparum and P. malariae circumsporozoite proteins. In 1986, 45%, 73%, 72%, 85%, and 59% of the persons in Asembo Bay had antibodies to the respective peptides. In Got Nyabondo, the rates were 44%, 67%, 56%, 36%, and 41%, respectively. All positivity rates increased with age. When next determined in 1987, the positivity rates and levels of reactivity were generally unchanged in Asembo Bay, but were decreased in Got Nyabondo.

Identification of malaria species by ELISA in sporozoite and oocyst infected Anopheles from western Kenya.

Enzyme-linked immunosorbent assays (ELISAs) for the circumsporozoite (CS) antigens of Plasmodium falciparum, P. malariae, and P. ovale were used to identify species of sporozoite and oocyst infections detected by dissection in Anopheles gambiae s.1. and An. funestus collected in western Kenya. ELISAs identified 92.5% of 1,113 salivary gland infections; Plasmodium species infections included 79.4% P. falciparum, 3.2% P. malariae, 1.7% P. ovale, and 2 or more Plasmodium species were detected in 15.7% of the Anopheles in which the species of parasite was identified. Identification was more likely with greater numbers of sporozoites observed in dissections, increasing from 65% ELISA positivity in mosquitoes with 1-10 sporozoites in their salivary glands to 96% in mosquitoes with over 1,000 sporozoites. ELISAs detected CS antigen in 66% of 294 Anopheles that by dissection had oocysts but uninfected salivary glands. Of 112 Anopheles with a single species of Plasmodium detected in the salivary glands, 29 (25.9%) had 1 or more additional species detected in the midgut, indicating a high potential for multiple infections. Similar proportions of Plasmodium species were found in An. gambiae s.1. and An. funestus.

Age-specific prevalence of antibody to a synthetic peptide of the circumsporozoite protein of Plasmodium falciparum in children from three villages in Kenya.

The presence of antibody to the repeating epitope of the circumsporozoite protein of Plasmodium falciparum was determined in children 1 month to 10 years old from three villages in western Kenya using the synthetic peptide (PNAN)5 in an enzyme-linked immunosorbent assay. The percentage of antibody-positive children increased with age and differed in the three villages. The village with the lowest percentage of antibody-positive children had the lowest percentage of infections as determined by detection of blood stage parasites. The villages also differed in the age at which antibody first appeared. In one village, only 12% of the children had antibody by the age of 5; while in the other two villages, 60% and 73% had antibody by 4 years of age.

Assessment of a synthetic DNA probe for Plasmodium falciparum in African blood specimens.

Synthetic DNA oligomers homologous to 21-base long repetitive sequences of Plasmodium falciparum DNA were labeled with 32P using T4 kinase, and were hybridized with purified DNA and with processed blood samples from Africa. The sequence PFR1, its antiparallel oligomer PFR1R, and PFR1 covalently attached to biotin hybridized similarly to P. falciparum DNA. One-microliter aliquots of blood from Zaire spotted on prewet nylon filters and hybridized with PFR1 gave detectable autoradiogram signals from samples with parasitemias as low as 1,000 parasites/mm3. Blood lysis and protein digestion followed by alkylation allowed dot-blot processing of larger aliquots of blood. After hybridization with PFR 1 and autoradiography, 26 samples were scored positive visually, compared with 34 scored positive by microscopy. The effective sensitivity for processed 10-microliter samples was about 500 parasites/mm3. Signals from hybridized probes were quantitated by liquid scintillation counting and densitometry, and were proportional to the amounts of purified P. falciparum DNA applied to the filter. Autoradiogram signals also were roughly proportional (correlation coefficient, r = 0.77) to the number of parasites/mm3 of blood from field samples as determined by microscopic examination.

In vivo response of Plasmodium falciparum to chloroquine in pregnant and non-pregnant women in Siaya District, Kenya.

Chemoprophylaxis using chloroquine (CQ) in suppressive doses has been recommended to protect pregnant women in malarious areas from the adverse effects of malaria during pregnancy. In a malaria-endemic area of western Kenya with CQ-resistant Plasmodium falciparum, we determined the prevalence and density of falciparum infection in gravid and nulligravid women and compared the in-vivo parasite response to CQ using two regimens: 25 mg/kg body weight (CQ25) divided over a period of three days (for high-density parasitaemias) and 5 mg/kg body weight (CQ5) weekly for 4 weeks (for low-density parasitaemias). P. falciparum infections were present in 102 (42%) of 244 pregnant women. A greater proportion of primigravidae were parasitaemic (68%) than nulligravidae (50%, P=0.02) or multigravidae (33%, P <10(-6)). Primigravidae showed a higher geometric mean parasite density. In the CQ25 treatment group, failure to clear parasites by day 7 was more common in primigravidae than nulligravidae (P=0.008) or multigravidae (P=0.15). In the CQ5 treatment group, primigravidae were more likely to show increasing parasite density than nulligravidae or multigravidae.In this area of Kenya, virtually all women in their first pregnancy are exposed to malaria and are at greatest risk for malaria infection; compared with other women, they show higher parasite densities and are least likely to respond to chloroquine treatment in areas of chloroquine resistance. Malaria control strategies might be targeted to this group of women, but we are pessimistic about the efficacy of weekly CQ5 where there is chloroquine resistance.

Sensitive analysis of blood for amodiaquine and three metabolites by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method using oxidative electrochemical detection has been developed for selective and sensitive quantification of the antimalarial drug amodiaquine and three of its metabolites in the blood of dosed individuals. The method requires only one extraction step and has detection limits of 1 ng/ml for amodiaquine and its metabolites desethylamodiaquine and bisdesethylamodiaquine and 3 ng/ml for 2-hydroxydesethylamodiaquine. Minor modification of the mobile phase preserves the chromatographic separation and allows ultraviolet spectroscopic detection, which, although appreciably less sensitive, permits monitoring of levels of amodiaquine and the three metabolites in blood and urine samples if an electrochemical detector is unavailable. Levels of amodiaquine and the three metabolites were determined for two volunteers undergoing a nine-week chemoprophylactic regimen in connection with travel to a malarious area. Data are included to compare the in vitro antimalarial activities against three strains of Plasmodium falciparum of amodiaquine and the three metabolites considered.

Time course of radiometric detection of positive blood cultures in childhood.

We have determined the time course of radiometric detection of microbial growth in 2348 positive blood culture specimens obtained at Wyler Children's Hospital during a 5-year interval. Overall 72 and 88% of isolates were detected within 48 and 72 hours after sampling, respectively. For pathogenic organisms aerobic detection was generally more rapid and more inclusive than anaerobic detection. At 48 hours of incubation the detection of six potential pathogens (Salmonella sp., Haemophilus influenzae, Group D streptococci, Neisseria meningitidis, coagulase-negative staphylococci, Candida sp.) was significantly delayed compared with detection of other pathogenic organisms recovered from blood. At 72 hours of incubation the detection rates remained less than 95% for H. influenzae, Staphylococcus aureus, Klebsiella sp., coagulase-negative staphylococci, Group D streptococci and Candida sp. These data should assist clinical decisions regarding duration of antibiotic therapy for the presumptive diagnosis of bacteremia in children.

Antibody response to preexposure human diploid-cell rabies vaccine given concurrently with chloroquine.

We conducted a randomized controlled trial to evaluate the antibody response of freshman veterinary students to intradermal human diploid-cell rabies vaccine administered concurrently with chloroquine, a drug frequently used for chemoprophylaxis against malaria. Fifty-one students who had not been vaccinated against rabies were enrolled: 26 received 300 mg of chloroquine base per week (the recommended dose for malaria prophylaxis); 25 did not receive chloroquine and served as controls. All subjects received 0.1 ml of rabies vaccine intradermally on days 0, 7, and 28. Chloroquine was administered weekly to the treatment group, beginning nine days before the first dose of vaccine and continuing until day 48. The mean rabies-neutralizing antibody titer for the chloroquine group was significantly lower than that for the control group on each day of testing--i.e., day 28 (P = 0.0094), day 49 (P = 0.0008), and day 105 (P = 0.0002)--although both groups had neutralizing antibody titers on days 49 and 105, according to the criteria of the Centers for Disease Control. The blood concentrations of chloroquine and desethylchloroquine (the major metabolite of chloroquine, which also has antimalarial properties) were negatively associated with log antibody titers. These results indicate that chloroquine taken in the dose recommended for malaria prophylaxis can reduce the antibody response to primary immunization with intradermal human diploid-cell rabies vaccine.

Recent trends in the importation of malaria caused by Plasmodium falciparum into the United States from Africa.

National malaria surveillance data were reviewed in an analysis of the epidemiological impact of the transmission of chloroquine-resistant Plasmodium falciparum in Africa on malaria in the United States. Between 1975 and 1983, P. falciparum infections acquired by U.S. citizens who visited East Africa, especially Kenya, increased 21-fold. Estimated attack rates for P. falciparum per 100,000 U.S. travelers to Kenya rose from 21.2 cases in 1977 to 83.3 cases in 1982, a rise suggesting that the increase in imported malaria was not due to increased travel. The percentage of reported cases in U.S. citizens with P. falciparum infections acquired in East Africa who indicated having used chloroquine prophylaxis increased from 22.2% in 1978 to 75.8% in 1983; in contrast, no change in the reported use of chloroquine prophylaxis was observed in those infected in West Africa during the same period. These results suggest that chloroquine can no longer be considered a highly effective drug for prevention of malaria caused by P. falciparum in U.S. travelers to East Africa.

Amodiaquine as a prodrug: importance of metabolite(s) in the antimalarial effect of amodiaquine in humans.

Existing analytical methods for assaying the 4-aminoquinoline antimalarial amodiaquine in body fluids are nonspecific and obscure the fact that little or no amodiaquine is present in the blood of dosed persons. We have isolated four metabolites of amodiaquine. The two major metabolites have been identified; one is desethylamodiaquine, and the other has been tentatively identified on the basis of proton nuclear magnetic resonance spectroscopy as 2-hydroxydesethylamodiaquine. We developed a reverse-phase high-performance liquid chromatographic (HPLC) method that separates the two major metabolites from each other and from amodiaquine, allowing separate quantification. The impact of these findings on in vitro sensitivity testing and blood analysis of persons dosed with amodiaquine is discussed.

In vivo and in vitro susceptibility to chloroquine of Plasmodium falciparum in Kinshasa and Mbuji-Mayi, Zaire.

From April to June 1983, combined in vivo and in vitro studies were conducted to assess the response to chloroquine of Plasmodium falciparum in Kinshasa and Mbuji-Mayi, Zaire. A total of 109 patients were treated with chloroquine, either as a single dose of 10 mg/kg or as a full dose of 25 mg/kg. All patients rapidly cleared their asexual parasitaemia, no recurrence being noted during the subsequent 3 weeks of follow-up. In the fourth week, recurrences were noted in 3 out of 66 patients treated with the full dose of chloroquine and in 10 out of 43 patients treated with the single dose. A total of 101 in vitro tests (30 macro tests, 39 micro tests, and 32 48-hour tests) were successfully performed with blood samples collected from 51 of these patients. Full sensitivity to chloroquine was demonstrated in all but 3 of the successful in vitro tests, the results from these 3 tests being contradicted either by alternative in vitro tests or by the corresponding in vivo findings. These investigations thus failed to detect chloroquine resistance at the level reported in East Africa or eastern Zaire (in Kivu).

Studies on the Uganda I/CDC strain of Plasmodium malariae in bolivian Aotus monkeys and various anophelines.

A strain of Plasmodium malariae (Uganda I/CDC) was isolated from an infant who had been infected via blood transfusion from a donor who had entered the United States 8 yr previously. After passage through a splenectomized chimpanzee, the parasite was studied in 29 splenectomized Aotus azarae boliviensis monkeys. Maximum parasitemias were higher in Aotus monkeys without previous Plasmodium infection than in Aotus monkeys with a history of P. vivax infection. Animals with a history of infection with both P. vivax and P. falciparum had lower maximum parasitemias than did monkeys in either of the 2 preceding groups. The highest rates of mosquito infection were most often obtained during the 10-day period just after the parasite count rose above 500/mm3 of blood. The most susceptible mosquito was Anopheles freeborni followed by An. stephensi, An. gambiae, An. dirus, and An. maculatus. Two attempts to transmit the Uganda I/CDC strain of P. malariae to other monkeys by sporozoite inoculation were unsuccessful.

Glucose-6-phosphate dehydrogenase deficiency in Southeast Asian refugees entering the United States.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an inherited disorder of red blood cell metabolism. Affected individuals may suffer a severe hemolytic crisis when treated with primaquine, an antimalarial. A survey of 966 male Southeast Asian refugees determined that 50 (5.2%) were G6PD-deficient. This prevalence suggests that a G6PD assay should be performed prior to primaquine therapy in this high-risk population.

Evaluation of amodiaquine treatment of chloroquine-resistant Plasmodium falciparum malaria on Zanzibar, 1982.

Amodiaquine, a 4-aminoquinoline which has been shown to be effective in treating infections with chloroquine-resistant strains of Plasmodium falciparum, was evaluated against chloroquine-resistant infections in children in Zanzibar, Tanzania, during July 1982. A 25-mg base/kg dosage of amodiaquine produced parasite clearance in 34 of 38 (89%) children in a mean of 2.8 days. When followed for 28 days, 15 of 38 (39%) children were completely cured of their infection as judged by the absence of renewed parasitemia. The parasite clearance rates produced by amodiaquine were significantly higher than those observed in a comparison group of children treated with 25 mg base/kg chloroquine. There was, however, no difference in the cure rates in the chloroquine and amodiaquine groups. Despite the enhanced parasite clearance rate, amodiaquine is not sufficiently more effective against Zanzibari strains of P. falciparum to replace chloroquine. Other alternative drugs must be evaluated to define the optimal malaria therapy regimen on Zanzibar.

Analysis of filter-paper-absorbed, finger-stick blood samples for chloroquine and its major metabolite using high-performance liquid chromatography with fluorescence detection.

Methodology has been developed to facilitate the collection, transport, and analysis of blood samples in studies of chloroquine absorption and metabolism. The method utilizes high-performance liquid chromatography (HPLC) with fluorescence detection to quantify chloroquine and its major metabolite, desethylchloroquine, in 100-microliters quantities of blood collected on filter paper. Detection limits are 5 ng/ml for both analytes. No loss of either analyte occurred from filter-paper-collected blood spots stored over a twelve-weeks' period at room temperature. Filter-paper-collected, finger-stick blood spots give values for each analyte comparable to corresponding determinations on venous, whole-blood samples. The HPLC mobile phase selected has general applicability to the separation of antimalarial drugs. The methodology permits effective assessment of chloroquine prophylaxis compliance and parasite drug resistance in remote, malaria-endemic regions.