Infected Anopheline Research Articles

Plasmodium sporozoites invade cells with targeted deletions in the LDL receptor related protein

Malaria infection is initiated when an infected anopheline mosquito injects sporozoites during a bloodmeal. After injection, sporozoites enter the circulation and rapidly invade hepatocytes [1], where they develop into exoerythrocytic forms (EEF). There is a large body of evidence (reviewed in [2]) suggesting that sporozoite arrest in the liver is mediated by the binding of the parasite’s major surface protein, the circumsporozoite protein (CS; [3]), to heparan sulfate proteoglycans (HSPGs) on the hepatocyte surface. CS binding to HSPGs is mediated by region II-plus, a highly conserved stretch of amino acids, containing a motif of basic and hydrophobic residues, which binds to the negatively-charged sulfate moieties of HSPG glycosaminoglycan (GAG) chains. Physiologic ligands for these hepatic HSPGs include chylomicron and very low density lipoprotein remnants [4]. Like Plasmodium sporozoites, these lipoprotein remnants are rapidly cleared from the circulation by the liver. Clearance of these lipoproteins is mediated by apolipoprotein (apo) E which, like CS, binds to HSPGs. We have previously shown, that remnant lipoproteins can competitively inhibit sporozoite invasion of HepG2 cells in vitro and can decrease sporozoite infectivity in vivo, suggesting that sporozoites and remnant lipoproteins compete for the same HSPG binding sites at the cell surface [5]. After their sequestration from the plasma, by hepatic HSPGs [6], remnant lipoproteins are internalized by one of two receptors found on the hepatocyte surface, the LDL receptor (LDLR) and the LDL receptor-related protein (LRP) [7– * Corresponding author. Tel.: +1-212-263-6818; fax: +1212-263-8116. E-mail address: photini.sinnis@med.nyu.edu (P. Sinnis)

Malaria transmission in the rural zone of Niakhar, Senegal

The anopheline bioecology and the malaria transmission were studied from January to December 1995 in three villages of the sahelian rural area of Niakhar, Senegal. This area of 29000 inhabitants, has been for several decades, a regional observatory for population and health. The three methods used for collecting mosquitoes were the collection at larval stages, the all night human biting collection, and the pyrethrum spray catch in houses during afternoons. The anophelines collected were, by numerical importance: Anopheles arabiensis, An. rufipes, An. gambiae, An. pharoensis, An. funestus and An. coustani. In the An. gambiae complex, An. arabiensis represented 97% of man biting females and 98% of half gravid resting females (difference not significant); the other reminding species of this complex was always An. gambiae. These two species belonging to the An. gambiae complex were responsible for the totality of the transmission. The anthropophilic index, obtained from half gravid indoor resting An. gambiae s.l., was 83%. The annual biting rate of An. gambiae s.l. varied from 512 to 1558 bites per man per night, depending on the villages. Vectors were observed all year long but their densities were low during the dry season. Vector population presented a notable increase due to the rains, with a maximum of about 10 bites per man per night in September or at the beginning of October; during September the biting rate represented 48% of the annual biting rate. The sporozoitic index of An. gambiae s.l., obtained by ELISA revealing the circumsporozoite protein, was 1.6% for human biting females and 1.8% for half-gravid resting females (difference not significant). Plasmodium falciparum was the only plasmodial species observed among infected anophelines. The annual transmission in the vo villages representative of the Niakhar area were 9 and 12 bites of infected anophelines per man, occurring mainly from August to October. In the third village, not representative of the area regarding permanent breeding places, the transmission was 26 bites of infected anopheline per man per year. These results were discussed in the Senegambian and sahelian contexts.

Transfection of Plasmodium: A new chapter in the molecular analysis of malaria

The development of a transfection system for the protozoan parasite Plasmodium falciparum hailed a breakthrough in understanding one of the world's most prevalent human pathogens. One hundred years after the discovery by Sir Ronald Ross that the etiological agent of malaria is transmitted to humans by the bite of infected Anopheline mosquitoes, we now have the tools at hand with which to investigate the biology and pathogenicity of P. falciparum, at the molecular level, in an attempt to develop sustainable means to control this important infectious disease. Here we review the recent progress facilitated by transfection in malariology and discuss the future opportunities this technique offers.

Intraspecific isozyme polymorphism of Anopheles gambiae in relation to environment, behavior, and malaria transmission in southwestern Burkina Faso.

A cellulose acetate electrophoresis system was used to study the isozyme polymorphism of the Anopheles gambiae complex in a rural village and a city in southwestern Burkina Faso. In both areas A. gambiae Giles was the dominant species (95%) whereas A. arabiensis Patton represented only 5%. Both species were separated readily by octanol dehydrogenase Odh and mannose phosphate isomerase (Mpi) even if they shared some alleles at these two loci. Polymorphism analysis (13 loci) at the intraspecific level of A. gambiae showed a significant difference between the specimens collected in the city from those collected in the village in their allelic and genotypic frequencies of isocitrate dehydrogenase-1 and malate dehydrogenase-1 and in their allelic frequencies for Mpi. No genetic difference was observed between the human biting A. gambiae collected inside or outside the houses in either the village or the city. The Plasmodium falciparum-infected A. gambiae differed from the noninfected ones in their allelic and genotypic frequencies at Mpi and acid phosphatase (Acp). A two-fold difference in infection rate was found for the genotype Mpi130/130 and Acp110/100 compared with other genotypes. However, infected anophelines were found in all genotypes that belonged to these two enzyme systems. Consequently, no refractory mechanism occurs in these natural populations.

Detection of Plasmodium falciparum sporozoites in naturally infected anopheline species using a fluorescein-labelled DNA probe

A dot blot hybridization method was developed to detect Plasmodium falciparum sporozoites in naturally infected mosquitoes. A fluorescein-labelled oligomer was used as a probe. Initial non-specific hybridization was found to correlate with the presence of blood in the mosquitoes. This was eliminated by allowing digestion of the engorged blood by keeping the mosquitoes in cages for 48 h before processing. The limit of detection of the hybridization assay was estimated to be about 500 sporozoites. The assay was evaluated on 198 indoor resting blood fed female Anopheles gambiae s.l mosquitoes collected from three malaria hypo- and meso-endemic areas in Ethiopia. An application of the polymerase chain reaction (PCR) amplifying a fragment of the K1-14 gene of P. falciparum was used as a reference method. P. falciparum sporozoites were detected in four specimens (2%) by hybridization assay and by PCR alike. The results of this study indicate that the hybridization method can be potentially valuable in large scale epidemiological studies for detection of P. falciparum sporozoites in naturally infected anopheline species.

Taxi rank malaria

EDITOR,--Malaria contracted during travel to a malarious zone may develop only after the traveller's return to an area free of the disease. This has led to doctors' practice of asking febrile patients, “Where have you been and when?” Malarious disease may also occur in people who have never visited a malarious area or been exposed to blood products infected with plasmodium. In north western Europe most such cases occur in the vicinity of international airports. These cases of “airport malaria” are acquired through the bites of infected anopheline mosquitos inadvertently imported by aircraft from endemic areas. …

Detection of Falciparum Malarial Forms in Naturally Infected Anophelines in Cameroon Using a Fluorescent Anti—25-kD Monoclonal Antibody

Anopheles gambiae s.s. and An. funestus were sampled in houses located in a Plasmodium falciparum-holoendemic site in southern Cameroon. The midguts of female mosquitoes in half-gravid or gravid stages of blood digestion were incubated with a fluorescent monoclonal antibody directed against the P. falciparum zygote/ookinete surface protein Pfs25 and examined using a fluorescent light microscope. Malarial forms were detected in 11.6% of the half-gravid mosquitoes and in 0.0% of the gravid ones (P = 0.012). No difference in infections or the occurrence of malarial forms between An. gambiae and An. funestus was observed. Overall, 127 malarial forms were counted and distributed among round forms, retorts, and ookinetes in 77.2%, 9.5%, and 13.4%, respectively. Round forms include macrogametes, activating microgametocytes, and zygotes. The mean number of malarial forms per infected midgut was 2.16 and the maximum number observed was 13. In four anophelines, round forms, retorts, and ookinetes were simultaneously observed. Sporozoite rates were 5.7% for An. gambiae and 3.8% for An. funestus. In the human population, the gametocyte index for P. falciparum was 38% with a mean density of 1.11 gametocytes per microliter of blood. Differences concerning malarial forms in mosquito midguts were observed between houses (range percentage = 4.7--21.3%; mean range of forms per positive anopheline = 1.1--3.1). In each house, relationships existed between infected vectors and the gametocyte reservoir of their inhabitants. The role in transmission of people with very low gametocytemia, approximately one per microliter, as a reservoir of falciparum malaria in highly endemic areas, is emphasized.

Laboratory methods for the conduct of experimental malaria challenge of volunteers

Experimental malaria challenge trials of volunteers are conducted to determine whether candidate sporozoite vaccines generate protective immunity against the bites of infected mosquitoes. The absence of patent erythrocytic-stage infection and clinical sequelae suggests sterile immunity among vaccinated volunteers. Sterile immunity is validated through laboratory studies that (1) demonstrate the capacity of erythrocytes and serum obtained from each volunteer before challenge to support parasite growth in vitro; (2) confirm parasitaemia by blood culture, and (3) evaluate each volunteer's blood for surreptitious use of antimalarial agents. The bites of experimentally infected laboratory-reared anophelines must elicit a 100% attack rate among non-immunized volunteers. This report presents guidelines for the laboratory methods used to validate the clinical findings during experimental Plasmodium falciparum anti-sporozoite vaccine efficacy trials.

Hypnozoites of Plasmodium Simiovale

Hypnozoites of Plasmodium simiovale were detected in liver biopsies from a rhesus monkey (Macaca mulatta) inoculated eight days previously with sporozoites from heavily infected anopheline mosquitoes. The tissue forms, 6 mu in diameter, were found within the cytoplasm of hepatic parenchymal cells by immunofluorescence and restained with Giemsa. This is the first report of latent, pre-erythrocytic stages from an ovale-type relapsing malaria.

Malaria parasites--discovery of the early liver form.

Infections of mammalian malaria parasites start when sporozoites from an infected anopheline mosquito are injected into the bloodstream of the host. The sporozoites enter the hepatocytes and become transformed into exoerythrocytic schizonts. Since the discovery of the primate parasite Plasmodium cynomolgi in monkey hepatocytes and the rodent parasite Plasmodium berghei in hamster hepatocytes, the ultrastructure of these stages has been extensively studied both in primate and rodent plasmodia. These observations relate only to the development of the exoerythrocytic schizont 25 h after sporozoite injection until the final maturation (of P. berghei) 50 h post-inoculation. Recently, we have studied the route of entry of sporozoites across the cellular lining of liver sinusoids and invasion of the liver parenchymal cells by using transmission electron microscopy. The results of these studies in combination with other physiological experiments strongly suggested that the sporozoite was initially harboured by the Kupffer cell, from which the parasite escaped into the neighbouring hepatocyte. The migration of sporozoites from liver sinusoids to hepatocytes can be achieved within a few minutes. We present here the first ultrastructural observations on the natural transformation of intrahepatocytic sporozoites into exoerythrocytic forms in vivo, using the rodent malaria parasite P. berghei in a laboratory host, the Brown Norway rat. These observations complete the search for the final link in the life cycle of malaria parasites.

Demonstration of hypnozoites in sporozoite-transmitted Plasmodium vivax infection.

Hypnozoites of two strains of the human relapsing malaria parasite, Plasmodium vivax, have been detected among maturing 7- and 10-day pre-erythrocytic schizonts in liver biopsies of chimpanzees infected by intravenous inoculation of sporozoites obtained from dissected salivary glands of heavily infected anopheline mosquitoes. As in the simian relapsing species, P. cynomolgi, the hypnozoites of P. vivax at 7 and 10 days are uninucleate forms of approximately 4-5 micrometers diameter, lying within the cytoplasm of individual hepatocytes. Their presence in this relapsing human species is added support for the hypnozoite theory of malarial relapse.

The Schizonticidal Activity of Hydroxynaphthoquinones against Plasmodium berghei Infections in Mice

Two decades ago 2-hydroxy-1,4-naphthoquinones were reported to have considerable suppressive and prophylactic activity against avian malaria. A few compounds were selected for clinical testing but were ineffective in human malarias presumably because of oxidation in the human body into inactive products. Recently homologs with longer n-alkyl side-chains than those of earlier compounds in the series have been synthesized and tested against rodent malarias. In four of the five series of 2-hydroxy-1,4-naphthoquinones reported in this study, peak activity occurred when the methylene groups adjacent to the alicyclic or terminal methyl group numbered 8 or 9. The higher members of the series have been demonstrated to possess not only schizonticidal activity at a dose level lower than that required for the older hydroxynaphthoquinones but also curative activity at high doses. The discovery of strains of Plasmodium falciparum refractory to either suppressive or therapeutic doses of 4-aminoquinolines (chloroquine, hydroxychloroquine, amodiaquine) and a-aminoacridines (quinacrine, propoquine) made reappraisal of malaria chemotherapy mandatory largely because of the known limitations of alternate drugs (chlorguanide, pyrimethamine, quinine, cycloguanil, primaquine, etc.). In an effort to discover new antimalarial drugs our attention was drawn to a group of 2-hydroxy-l,4-naphthoquinone compounds synthesized by Fieser and his associates at Harvard University's Chemical Laboratory as a part of the antimalarial survey conducted on a national scale during World War II. Two of these naphthoquinones (M-1916 and M-285) were shown to possess suppressive and curative effects against avian plasmodia (P. lophurae and P. gallinaceum), but they did not prove to have significant effects against P. vivax and P. falciparum infections of humans (Fieser et al., 1948a). These and other related naphthoquinone antimalarials received scant attention at that time because of the marked suppressive and therapeutic efficacy of 4aminoquinoline antimalarials at well tolerated dose levels. After the discovery of rodent plasmodial infections (P. berghei, P. vinckei, P. chabaudi), it became possible to screen drugs on a wide scale against mammalian rather than avian plasmodia. Several of the old and a few of the more recently synthesized hydroxynaphthoquinone antimalarials (Fieser et al., 1967a, Received for publication 12 March 1968. b) were screened and evaluated against bloodinduced rodent infections and were found to be effective following oral administration. The discovery by Yoeli and Most in 1960 of means for facilitating the transfer of P. berghei through the bites of infected anophelines under laboratory conditions made it possible for us to test the efficacy of the new hydroxynaphthoquinone derivatives against sporozoiteinduced as well as blood-induced P. berghei infections in mice. This paper describes the antimalarial screening procedures used and presents the activity of several compounds belonging to each of the various series of 3-substituted alkyl-2-hydroxy-1,4-naphthoquinones against blood-induced P. berghei infections in mice. A few selected derivatives from the various series of hydroxynaphthoquinones were administered to mice infected by injection of sporozoites of P. berghei. The details of these tests are the basis for a separate paper. MATERIALS AND METHODS Rodent plasmodia Four strains of Plasmodium berghei (NYU-2, NK-65, KSP-11, and subspecies yoelii), two other rodent plasmodia (P. vinckei and P. chabaudi), and a strain of P. berghei maintained under chloroquine pressure were used in our studies. In this paper the results of screening against two strains of P. berghei (NYU-2 and NK-65) will be presented. The NYU-2 strain was isolated on 6 January 1959 by Dr. Van Braeckel from infected salivary glands of Anopheles dureni caught in the vicinity of Kasapa (near Elizabethville, Congo). Dr. Yoeli of New York University obtained the strain from Professor Jadin of Belgium on 20 February 1959. Although originally a vigorous gametocyte producer, it gradually lost this ability through numer-

A new approach to the measurement of residual transmission in a malaria control scheme in East Africa

1. 1) In the 4th year of protection in a malaria control scheme, despite the absence of infected anophelines and a very low infant parasite rate, infections with P. falciparum were still fairly common in children and adolescents. 2. 2) After a preliminary trial, which encouraged belief in the adequacy of the doses used, a group of people were treated with drugs to clear them of infections with P. falciparum. When examined 4 months later all those found infected on the first examination were clear of parasites while, conversely, a number who were uninfected earlier had a parasitaemia. These results were interpreted as evidence of recent transmission, although certainty must await more complete knowledge of the biology of P. falciparum infections in partially immune Africans, and the reaction of such infections to small doses of drugs. 3. 3) Doubt is thrown on the infant parasite rate as the most satisfactory index of malaria transmission in eradication schemes and an alternative method suggested.