Phuc Nguyen-Dinh

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.

RAPID SPONTANEOUS POSTPARTUM CLEARANCE OF PLASMODIUM FALCIPARUM PARASITAEMIA IN AFRICAN WOMEN

During pregnancy susceptibility to Plasmodium falciparum infection increases with serious implications for the pregnancy. 60 symptom free otherwise healthy women infected with P. falciparum who presented for delivery were studied. Giemsa stained peripheral blood smears were obtained before delivery and 4 to 48 hr. after delivery to quantify asexual parasites. Antimalarial drugs were not given until 48 hrs. after delivery. 15 of 17 women in Kinshasa Zaire cleared parasitemia within 24 hours of delivery and remained parasite free on followup. 35 of 43 women (81%) in Mangochi Malawi cleared their parasitemia within 24 hours and 95% by 48 hours. There was no correlation between parasite clearance and predelivery parasite burden age parity or presence of antimalarial drugs in the blood. In 4 women seen with P. malariae infection parasitemia did not clear by 48 hours after delivery. Most symptom free women infected with P. falciparum cleared peripheral parasites rapidly and spontaneously after delivery without antimalarial drugs. This clearance may be mechanical due to removal of the large parasite load in the placenta or may be due to immunological/hormonal factors at the time of delivery.

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.

LACK OF EFFICACY OF PYRIMETHAMINE PROPHYLAXIS IN PREGNANT NIGERIAN WOMEN

To evaluate the efficacy of pyrimethamine on the blood stage (suppressive prophylaxis) and liver stage (causal prophylaxis) of Plasmodium falciparum in pregnant women, in vivo and in vitro field studies were conducted in Ilorin, Nigeria, from Jan 1 to June 30, 1988. For pregnant women with P falciparum infections who received 25 mg of pyrimethamine weekly for suppressive prophylaxis, 67% (59/88) of in vivo and 60% (6/10) of in vitro tests showed pyrimethamine resistance. A second group of parasitaemic and parasite-free pregnant women was enrolled to evaluate the efficacy of pyrimethamine as a primary tissue schizonticide; after receiving a curative dose of chloroquine (25 mg/kg), half the women were given 25 mg of pyrimethamine weekly and half received no prophylaxis. Parasitologic failure rates did not differ between the pyrimethamine-treated (8/34) and the control (11/37) groups during the 16-week follow-up. Thus, pyrimethamine is not effective for suppressive or causal prophylaxis in pregnant women in Ilorin.

Sensitive high-performance liquid chromatographic analysis for chloroquine in body fluids application to studies of drug resistance in plasmodium falciparum

A high-performance liquid chromatographic method has been developed for the sensitive determination of chloroquine in body fluids. THe method has been applied to quality-control assay of World Health Organization (WHO) In-Vitro, Macro-Test Kits for the assessment of susceptibility of Plasmodium falciparum to chloroquine. Experiments utilizing [14C] chloroquine demonstrated that water was not capable of efficiently desorbing chloroquine from the inside surfaces of kit vials. The addition of blood to the vials effectively desorbs chloroquine. Subsequent addition of the blood to aqueous base followed by hexane extraction permits quantitation by reversed-phase, ion-pair high-performance liquid chromatography utilizing ultraviolet detection at 344 nm. The method is capable of determining as little as 20 ng of chloroquine per vial. This method, utilizing the methyl ether of 9-anthra cenemethanol as internal standard, can quantify chloroquine in 1 ml of blood or urine with a minimum detection limit of 20 ppb (ng/ml). Measurement of blood levels of chloroquine in persons contracting falciparum malaria while following a prophylactic regimen complements in-vitro drug susceptibility measurements in characterizing resistant strains of the parasite.

Adaptation of a Strain of Plasmodium vivax from Mauritania to New World Monkeys and Anopheline Mosquitoes

A strain of Plasmodium vivax from Mauritania was adapted to develop in Aotus lemurinus griseimembra, Aotus nancymai, Saimiri boliviensis, and hybrid Aotus monkeys. Infections were induced via the inoculation of sporozoites dissected from the salivary glands of Anopheles gambiae, Anopheles freeborni, and Anopheles stephensi mosquitoes or the intravenous passage of infected erythrocytes. Infections in 3 A. lemurinus griseimembra monkeys readily infected mosquitoes. Four lines of the Mauritania parasites have been stored frozen for further reference.

OBSERVATIONS ON THE INFECTIVITY OF TWO STRAINS OF PLASMODIUM VIVAX FROM VIETNAMESE REFUGEES TO AOTUS MONKEYS AND ANOPHELINE MOSQUITOES

Two strains of Plasmodium vivax (NAM and ONG) were isolated from Vietnamese refugees and established in splenectomized Aotus monkeys from Colombia and Bolivia. Mosquito infections were readily obtained from animals with no prior malarial experience or with a history of infection with P. falciparum only. Those animals with previous infections with P. vivax supported only low parasitemias, and the mosquito infections were minimal. Complete development of the sporogonic cycle was obtained with all species of mosquitoes tested. The most susceptible mosquito was An. dirus. Other mosquitoes readily infected with these strains were An. culicifacies, An. maculatus, An. gambiae, An. stephensi, and the two North American species, An. freeborni and An. quadrimaculatus. Transmission from one monkey to another was obtained via the bites of infected An. dirus, An. stephensi, and An. maculatus mosquitoes.

STUDIES ON THE INDOCHINA I/CDC STRAIN OF PLASMODIUM FALCIPARUM IN COLOMBIAN AND BOLIVIAN AOTUS MONKEYS AND DIFFERENT ANOPHELINES

The Indochina I/CDC strain of Plasmodium falciparum was isolated from a physician returning to the United States after working in the refugee camps along the Thailand-Kampuchean border. The strain was established in splenectomized Aotus monkeys from Colombia after being grown in vitro for 50 days. During the first three passages in Colombian monkeys, the parasites were not infective to Bolivian Aotus monkeys. After six intervening passages in Saimiri sciureus monkeys, the parasites produced high parasitemias in both Colombian and Bolivian Aotus, but gametocytes were no longer produced. Mosquito infections were obtained only during the first three passages in the Colombian monkeys. The most susceptible mosquito was Anopheles freeborni, followed by An. dirus, An. stephensi, An. maculatus, An. culicifacies, and, rarely, An. gambiae. Sporozoites were found in the salivary glands of the An. freeborni, An. dirus, An. stephensi, and An. maculatus.

Characterization of Plasmodium falciparum cloned lines with respect to gametocyte production in vitro, infectivity to Anopheles mosquitoes, and transmission to Aotus monkeys

The production of gametocytes in vitro and their subsequent infectivity to mosquitoes by 3 cloned lines of Plasmodium falciparum were studied. 2 of the cloned lines, Honduras I-clone B3 and Indochina III-clone W2, produced mature gametocytes (stage V) that were infective to Anopheles mosquitoes. The third clone, Sierra Leone I-clone D6, produced gametocytes, the majority of which did not develop beyond stage III. Fully mature gametocytes of Sierra Leone I-clone D6 were not infective to mosquitoes. Sporozoites collected from An. freeborni infected with Honduras I-clone B3 were used in transmission studies. Two of three Aotus monkeys were infected after prepatent periods of 19 and 20 d, respectively. This study supports previous reports that cloned lines of P. falciparum contain the full genetic capacity to produce morphologically mature gametocytes. The transmission to Aotus monkeys has also conclusively established that biologically competent gametocytes of both sexes are produced by clones.

Plasmodium fragile: Detection of a ring-infected erythrocyte surface antigen (RESA)

A ring-infected erythrocyte surface antigen (RESA) has been detected by modified immunofluorescence assay in erythrocytes infected with the simian malaria parasite, Plasmodium fragile. This RESA, of Mr 95,000, shares many characteristics with the RESA initially found in the human malaria parasite P. falciparum. Both antigens are found in the membrane of erythrocytes infected with young asexual parasite stages, in merozoite-enriched preparations, and in parasite culture supernatant. Since the RESA of P. falciparum has been shown to confer protective immunity and since P. fragile infection of rhesus monkeys mimics P. falciparum infection in humans, the finding of a RESA in P. fragile underlines the importance of this species as an animal model for antimalarial vaccines.