Protocol for Plasmodium falciparum infections in mosquitoes and infection phenotype determination

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.     

Inhibition of the in vitro growth of Plasmodium falciparum. I. The effects of immune serum and purified immunoglobulin from owl monkeys.

Sera from Aotus sp. monkeys (karyotypes II, III, and IV) which were immune to Plasmodium falciparum have been used to inhibit the in vitro growth of this human malaria parasite. Culture conditions used for the assays allowed 50- to 100-fold increases in the number of A+ erythrocytes infected in a 96-hr period in control cultures. Although normal monkey serum did not support growth as well as normal human serum, mixtures of normal monkey and human serum were found that did. Compared to such controls, as little as 3.5% immune monkey serum was found to cause approximately 56% inhibition in 4 days (2 replicative cycles). Purified globulin from immune monkeys inhibited 40% at 2 mg/ml and 75% at 7 mg/ml after a single replicative cycle. These data suggest that serum antibody is likely to play a major role in providing Aotus monkeys with protective immunity to P. falciparum.     

Reduced Susceptibility of Plasmodium falciparum to Artesunate in Southern Myanmar

Background

Plasmodium falciparum resistance to artemisinins, the first line treatment for malaria worldwide, has been reported in western Cambodia. Resistance is characterized by significantly delayed clearance of parasites following artemisinin treatment. Artemisinin resistance has not previously been reported in Myanmar, which has the highest falciparum malaria burden among Southeast Asian countries.

Methods

A non-randomized, single-arm, open-label clinical trial of artesunate monotherapy (4 mg/kg daily for seven days) was conducted in adults with acute blood-smear positive P. falciparum malaria in Kawthaung, southern Myanmar. Parasite density was measured every 12 hours until two consecutive negative smears were obtained. Participants were followed weekly at the study clinic for three additional weeks. Co-primary endpoints included parasite clearance time (the time required for complete clearance of initial parasitemia), parasite clearance half-life (the time required for parasitemia to decrease by 50% based on the linear portion of the parasite clearance slope), and detectable parasitemia 72 hours after commencement of artesunate treatment. Drug pharmacokinetics were measured to rule out delayed clearance due to suboptimal drug levels.

Results

The median (range) parasite clearance half-life and time were 4.8 (2.1–9.7) and 60 (24–96) hours, respectively. The frequency distributions of parasite clearance half-life and time were bimodal, with very slow parasite clearance characteristic of the slowest-clearing Cambodian parasites (half-life longer than 6.2 hours) in approximately 1/3 of infections. Fourteen of 52 participants (26.9%) had a measurable parasitemia 72 hours after initiating artesunate treatment. Parasite clearance was not associated with drug pharmacokinetics.

Conclusions

A subset of P. falciparum infections in southern Myanmar displayed markedly delayed clearance following artemisinin treatment, suggesting either emergence of artemisinin resistance in southern Myanmar or spread to this location from its site of origin in western Cambodia. Resistance containment efforts are underway in Myanmar.

Trial Registration

Australian New Zealand Clinical Trials Registry ACTRN12610000896077     

Adaptation of Plasmodium vivax to growth in owl monkeys (Aotus nancymai)

The purpose of this study was reactivation and adaptation of a strain of Plasmodium vivax to Aotus nancymai monkeys. A need arose for malarial parasites for use in serologic and molecular studies and for teaching slides. This particular strain of parasite had been characterized previously as producing high-density parasitemia in splenectomized New World monkeys and therefore represented a good candidate for reactivation. P. vivax (Vietnam II), isolated in 1970, was reactivated after adaptation in Aotus lemurinus griseimembra monkeys nearly 33 years earlier and adapted to A. nancymai monkeys. Passage was achieved by intravenous inoculation of parasite blood stages into splenectomized A. nancymai monkeys. Parasitemia was determined by analyzing daily blood smears stained with Giemsa. Maximum parasite counts ranged from 10,630 to 94,000 parasites/microl; the mean maximum parasite count for the four animals was 39,565 parasites/microl. Parasite counts of > 10,000/microl were maintained for 2 to 64 days. After only three passages of the parasite, attempts to reactive were successful. A. nancymai proved a suitable animal model for the recovery of this parasite. In conclusion, successful reactivation and adaptation of this parasite offers the capability to perform a series of diagnostic, immunologic, and molecular studies as well as to provide otherwise potentially unavailable teaching materials to healthcare professionals.     

Reproducible infection of intact Aotus lemurinus griseimembra monkeys by Plasmodium falciparum sporozoite inoculation

Aotus lemurinus griseimembra is considered one of the best nonhuman primate species for malarial studies because of its susceptibility to infection by Plasmodium falciparum asexual blood stages. However, reproducible transmission of infective P. falciparum sporozoites by mosquito inoculation has been difficult to achieve even in splenectomized monkeys. Characterization of an Aotus-P. falciparum cyclical transmission model has become a top priority as a result of the significant progress toward the development of preerythrocytic malaria vaccines. Herein, we describe a reproducible model developed using intact A. lemurinus griseimembra monkeys intravenously inoculated with sporozoites from a monkey-adapted P. falciparum (Santa Lucia) strain and a wild Falciparum-Cali-Colombia-4 (FCC-4) strain. Sporozoites were obtained by salivary gland dissection of laboratory-reared Anopheles albimanus mosquitoes. Parasitemia was monitored by thick-smear microscopy, parasite lactate dehydrogenase (pLDH) determination, and mosquito xenodiagnosis. The last method proved to be the most sensitive method for monitoring parasitemias. Infection with the Santa Lucia strain showed a mean prepatent period of 16 days (range 6-21 days), whereas infection with the wild FCC-4 strain resulted in a 24-day prepatent period. Mean peak parasite density was approximately 900 parasites/microliter for both parasite strains. The prepatent period, the peak of parasitemia, and the duration of patency were independent of the size of the sporozoite inoculum and the presence of spleen in the host. This model is being successfully used to test the protective efficacy of P. falciparum preerythrocytic vaccine candidates.     

Anemia and antibodies to the 19-kDa fragment of MSP1 during Plasmodium falciparum infection in Aotus monkeys

To determine whether antibodies to the 19-kDa fragment of merozoite surface protein 1 (MSP1(19)) help to control blood-stage Plasmodium falciparum infection, we performed a rechallenge experiment of previously infected Aotus monkeys. Monkeys previously exposed to the FVO strain of P. falciparum that did or did not develop high antibody titers to MSP1(19) and malaria-na?ve monkeys were challenged with erythrocytes infected with the same strain. Prepatent periods were prolonged in previously infected monkeys compared with malaria-na?ve monkeys. Previously infected monkeys with preexisting anti-MSP1(19) antibodies showed low peak parasitemias that cleared spontaneously. Previously infected monkeys that had no or low levels of pre-existing anti-MSP1(19) antibodies also showed low peak parasitemias, but because of low hematocrits, all of these animals required treatment with mefloquine. All previously malaria-na?ve animals were treated because of high parasitemias. The results of this study suggest that antibody to the 19-kDa carboxy-terminal fragment of MSP1 plays a role in preventing the development of anemia, an important complication often associated with malaria.     

Susceptibility of Macaca fascicularis monkeys from Mauritius to different species of Plasmodium.

Macaca fascicularis monkeys from Mauritius were shown to be susceptible via sporozoite inoculation to 7 species of Plasmodium (P. fragile, P. coatneyi, P. gonderi, P. inui, P. cynomolgi, P. knowlesi, and P. fieldi), indigenous to macaques in southeastern Asia. Four monkeys were sequentially infected with different species of Plasmodium to determine maximum and course of parasitemia. In 2 nonsplenectomized monkeys, P. fragile developed maximum parasite counts of only 134 and 155/microliters. For Plasmodium knowlesi, a parasite that is life-threatening to rhesus monkeys, maximum parasite counts were 4,278 and 7,440/microliters. Plasmodium coatneyi developed to what must be considered as moderate levels. After animals underwent splenectomy, parasite counts of P. coatneyi were 58,280, 89,094, 4,464, and 43,524/microliters. The maximum parasite counts for P. gonderi (13,508 and 21,576/microliters) and P. fieldi (1,767 and 17,836/microliters) were lower than would be expected in M. mulatta. In 2 monkeys that developed patent parasitemia with P. inui, the maximum parasite counts (95,046 and 728,748/microliters) indicated that this parasite may be the best adapted species for development in these animals once infection is established. Finally, the reinfection of 2 monkeys with P. cynomolgi suggested that some animals may be basically more resistant than others, whether splenectomized or not, to the production of high-density parasitemia.     

Susceptibility of Anopheles campestris-like and Anopheles barbirostris species complexes to Plasmodium falciparum and Plasmodium vivax in Thailand

Nine colonies of five sibling species members of Anopheles barbirostris complexes were experimentally infected with Plasmodium falciparum and Plasmodium vivax. They were then dissected eight and 14 days after feeding for oocyst and sporozoite rates, respectively, and compared with Anopheles cracens. The results revealed that Anopheles campestris-like Forms E (Chiang Mai) and F (Udon Thani) as well as An. barbirostris species A3 and A4 were non-potential vectors for P. falciparum because 0% oocyst rates were obtained, in comparison to the 86.67-100% oocyst rates recovered from An. cracens. Likewise, An. campestris-like Forms E (Sa Kaeo) and F (Ayuttaya), as well as An. barbirostris species A4, were non-potential vectors for P. vivax because 0% sporozoite rates were obtained, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. barbirostris species A1, A2 and A3 were low potential vectors for P. vivax because 9.09%, 6.67% and 11.76% sporozoite rates were obtained, respectively, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. campestris-like Forms B and E (Chiang Mai) were high-potential vectors for P. vivax because 66.67% and 64.29% sporozoite rates were obtained, respectively, in comparison to 90% sporozoite rates recovered from An. cracens.     

The origin of antigenic diversity in Plasmodium falciparum

Most studies of genetic variability of Plasmodium falciparum have focused on protein antigens and the genes that encode them. The consensus is that populations exhibit high levels of genetic polymorphism, most notably the genes encoding surface proteins of the merozoite (Msp1, Msp2) and the sporozoite (Csp). The age and derivation of this variation is a subject that warrants further careful consideration, as discussed here by Stephen Rich, Marcelo Ferreira and Francisco Ayala.     

Extensive polymorphism and ancient origin of Plasmodium falciparum

DNA sequence data reveal extensive polymorphism in the virulent, human malaria parasite Plasmodium falciparum. The extent of polymorphism at apparently neutral-evolving loci points to a common ancestor for this species that is no more recent than approximately 150,000-200,000 years ago. In addition, there is evidence of balanced polymorphisms at certain antigen-encoding loci, some of which have been maintained for millions of years. Thus, we can reject the hypothesis that this species underwent a recent extreme bottleneck (i.e. one in which the population was reduced to a single haploid genotype). However, it is possible that less-severe bottlenecks have occurred.     

Susceptibility of in vitro cultured Plasmodium falciparum to chloroquine and combination of sulfadoxine and pyrimethamine

Keittivuti B., Keittivuti A. and Panichacheewakul P. 1982. Susceptibility of in vitro cultured Plasmodium falciparum to chloroquine and combinations of sulfadoxine and pyrimethamine. International Journal for Parasitology12: 383–387. The culture of P. falciparum in RPMI 1640 media in vitro was used for testing antimalarial drugs on blood from 11 patients at the Malaria Eradication Center, Phabuddhabat District, Saraburi Province, Thailand. Chloroquine in concentrations of 1, 3 and 4 nmol, combinations of sulfadoxine and pyrimethamine at 0.05 mg + 2.5 μg, 0.10 mg + 5 μg and 0.20 mg + 10 μg respectively and 1.4 μg sodium hydroxide/ml of blood were used. The organisms in the culture were resistant to the action of the combinations of sulfadoxine and pyrimethamine but were susceptible to chloroquine particularly at the 3 and 4 nmol levels. The in vitro test system used offers a valuable tool for studying the activity of antimalarial drugs.     

Platelet kinetics and other hematological profiles in experimental Plasmodium falciparum infection: a comparative study between Saimiri and Aotus monkeys.

Levels of platelets and other hematological values were monitored in 21 Saimiri and 12 Aotus monkeys over a period of three weeks post-infection with monkey-adapted Indochina CDC-1 strain of Plasmodium falciparum. In both Saimiri sciureus boliviensis and Aotus nancymai karyotype-1 monkeys the severest thrombocytopenia was observed at 14 days post-infection coinciding with peak parasitemia, neutropenia, lymphocytosis, and anemia associated with severe hemoglobinemia and elevated fibrinogen degeneration products(FDP's). MCH and MCV profiles in Aotus monkeys decreased with ascending parasitemia. In contrast, these parameters in Saimiri were characterized by a significant compensatory increase correlating with parasitemia. In general, thrombocytopenia was one of the earliest clinical manifestations of the infection with the platelets returning to normal levels shortly after peak parasitemia at 14 days. Platelet kinetics had a strong correlation with hematologic and parasitologic values in the Aotus model. No consistent associations were observed between platelet kinetics and other parameters in the Saimiri model. These data indicate that the Aotus model for malaria is more predictable than the Saimiri. Further, platelet turnover rates and recovery provide a useful prognostic parameter during malaria infection. The results are discussed in relation to the value of the two species of monkeys as models for the pathogenesis of human malaria.     

Evolution of Plasmodium and the recent origin of the world populations of Plasmodium falciparum

We have investigated the evolution of Plasmodium parasites by analyzing DNA sequences of several genes. We reach the following conclusions: (1) The four human parasites, P. falciparum, P. malariae, P. ovale, and P. vivax are very remotely related to each other, so that their evolutionary divergence predates the origin of the hominids; several of these parasites became associated with the human lineage by lateral transfer from other hosts. (2) P. falciparum diverged from P. reichenowi about 8 million years ago, consistently with the time of divergence of the human lineage from the apes; a parsimonious inference is that falciparum has been associated with humans since the origin of the hominids. (3) P. malariae is genetically indistinguishable from P. brasilianum, a parasite of New World monkeys; and, similarly. (4) P. vivax is genetically indistinguishable from the New World monkey parasite P. simium. We infer in each of these two cases a very recent lateral transfer between the human and monkey hosts, and explore alternative hypotheses about the direction of the transfer. We have also investigated the population structure of P. falciparum by analyzing 10 genes and conclude that the extant world populations of this parasite have evolved from a single strain within the last several thousand years. The extensive polymorphisms observed in the highly repetitive central region of the Csp gene, as well as the apparently very divergent two classes of alleles at the Msa-1 gene, are consistent with this conclusion.     

Infection of Peruvian Aotus nancymai monkeys with different strains of Plasmodium falciparum, P. vivax, and P. malariae

Aotus nancymai (karyotype I) monkeys from Peru were studied for their susceptibility to infection with Plasmodium falciparum, P. vivax, and P. malariae. Three strains of P. falciparum (Santa Lucia from El Salvador, Indochina I/CDC from Thailand, and Uganda Palo Alto) were inoculated into 38 monkeys. The results indicated that this species of Aotus monkey is highly susceptible to infection. The Uganda Palo Alto and the Santa Lucia strain parasites appear to be the most useful for immunologic and chemotherapeutic studies. Five strains of P. vivax (Chesson, ONG, Vietnam Palo Alto, Salvador I, and Honduran I/CDC) were inoculated into 28 monkeys. The Vietnam Palo Alto strain produced the highest level parasitemias ranging from 23,800 to 157,000/mm3. Mosquito infections were obtained with the ONG, Chesson, and Salvador I strains. Two out of 6 attempts to transmit P. vivax via sporozoite inoculation to splenectomized monkeys were successful with prepatent periods of 39 and 57 days. Five monkeys were infected with the Uganda I/CDC strain of P. malariae. Maximum parasitemias ranged from 10 to 5,390/mm3.     

The karyotype of Plasmodium falciparum determined by ultrastructural serial sectioning and 3D reconstruction

The karyotype of the erythrocytic stages of Plasmodium falciparum was determined to be 14 by counting the number of kinetochores in the mitotic spindle of young schizonts. Fourteen pairs of kinetochores were identified in 3-dimensional reconstructions of the spindle derived from serial longitudinal and transverse sections. Kinetochores were ovoid (45 X 25 nm) in transverse sections, measured 100 nm in longitudinal section, and were heptalaminate in structure. A pair of spindle microtubules passed through each kinetochore. In schizonts at anaphase, each pole of the mitotic spindle consisted of paired kinetic centers inserted on the nuclear membrane. The kinetic centers resembled nuclear pores, but were more electron dense and were associated with spherical masses of an electron-dense cytoplasmic material.