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.     

Reduced variation around drug-resistant dhfr alleles in African Plasmodium falciparum

We have measured microsatellite diversity at 26 markers around the dhfr gene in pyrimethamine-sensitive and -resistant parasites collected in southeast Africa. Through direct comparison with diversity on sensitive chromosomes we have found significant loss of diversity across a region of 70 kb around the most highly resistant allele which is evidence of a selective sweep attributable to selection through widespread use of pyrimethamine (in combination with sulfadoxine) as treatment for malaria. Retrospective analysis through four years of direct and continuous selection from use of sulfadoxine-pyrimethamine as first-line malaria treatment on a Plasmodium falciparum population in KwaZulu Natal, South Africa, has revealed how recombination significantly narrowed the margins of the selective sweep over time. A deterministic model incorporating selection coefficients measured during the same interval indicates that the transition was toward a state of recombination-selection equilibrium. We compared loss of diversity around the same resistance allele in two populations at either extreme of the range of entomological inoculation rates (EIRs), namely, under one infective bite per year in Mpumalanga, South Africa, and more than one per day in southern Tanzania. EIRs determine effective recombination rates and are expected to profoundly influence the dimensions of the selective sweep. Surprisingly, the dimensions were broadly consistent across both populations. We conclude that despite different recombination rates and contrasting drug selection histories in neighboring countries, the region-wide movement of resistant parasites has played a key role in the establishment of resistance in these populations and the dimensions of the selective sweep are dominated by the influence of high initial starting frequencies.     

A Randomised Controlled Trial of Artemether-Lumefantrine Versus Artesunate for Uncomplicated Plasmodium falciparum Treatment in Pregnancy

Background

To date no comparative trials have been done, to our knowledge, of fixed-dose artemisinin combination therapies (ACTs) for the treatment of Plasmodium falciparum malaria in pregnancy. Evidence on the safety and efficacy of ACTs in pregnancy is needed as these drugs are being used increasingly throughout the malaria-affected world. The objective of this study was to compare the efficacy, tolerability, and safety of artemether-lumefantrine, the most widely used fixed ACT, with 7 d artesunate monotherapy in the second and third trimesters of pregnancy.

Methods and Findings

An open-label randomised controlled trial comparing directly observed treatment with artemether-lumefantrine 3 d (AL) or artesunate monotherapy 7 d (AS7) was conducted in Karen women in the border area of northwestern Thailand who had uncomplicated P. falciparum malaria in the second and third trimesters of pregnancy. The primary endpoint was efficacy defined as the P. falciparum PCR-adjusted cure rates assessed at delivery or by day 42 if this occurred later than delivery, as estimated by Kaplan-Meier survival analysis. Infants were assessed at birth and followed until 1 y of life. Blood sampling was performed to characterise the pharmacokinetics of lumefantrine in pregnancy. Both regimens were very well tolerated. The cure rates (95% confidence interval) for the intention to treat (ITT) population were: AS7 89.2% (82.3%–96.1%) and AL 82.0% (74.8%–89.3%), p = 0.054 (ITT); and AS7 89.7% (82.6%–96.8%) and AL 81.2% (73.6%–88.8%), p = 0.031 (per-protocol population). One-third of the PCR-confirmed recrudescent cases occurred after 42 d of follow-up. Birth outcomes and infant (up to age 1 y) outcomes did not differ significantly between the two groups. The pharmacokinetic study indicated that low concentrations of artemether and lumefantrine were the main contributors to the poor efficacy of AL.

Conclusion

The current standard six-dose artemether-lumefantrine regimen was well tolerated and safe in pregnant Karen women with uncomplicated falciparum malaria, but efficacy was inferior to 7 d artesunate monotherapy and was unsatisfactory for general deployment in this geographic area. Reduced efficacy probably results from low drug concentrations in later pregnancy. A longer or more frequent AL dose regimen may be needed to treat pregnant women effectively and should now be evaluated. Parasitological endpoints in clinical trials of any antimalarial drug treatment in pregnancy should be extended to delivery or day 42 if it comes later. Trial Registration: Current Controlled Trials ISRCTN86353884     

High levels of antibodies to Plasmodium falciparum liver stage antigen-1 in naturally infected individuals in Myanmar

Plasmodium falciparum liver stage antigen-1 (PfLSA-1) is one of the few antigens expressed exclusively in liver stage parasites. In this study, we evaluated the antibody responses against recombinant PfLSA-1 in naturally infected individuals in Myanmar. High levels of antibody responses (70.7%) were detected in 82 serum samples from 116 infected individuals, and IgG responses to PfLSA-1 principally composed of responses of IgG1 and IgG3 subclasses. These results show that PfLSA-1 elicits effective antibody responses in individuals infected with P. falciparum, and thus it could be not only an attractive candidate protein for vaccine development, but also a useful antigen for serodiagnosis of the infection.     

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.     

A Genome Wide Association Study of Plasmodium falciparum Susceptibility to 22 Antimalarial Drugs in Kenya

Background

Drug resistance remains a chief concern for malaria control. In order to determine the genetic markers of drug resistant parasites, we tested the genome-wide associations (GWA) of sequence-based genotypes from 35 Kenyan P. falciparum parasites with the activities of 22 antimalarial drugs.

Methods and Principal Findings

Parasites isolated from children with acute febrile malaria were adapted to culture, and sensitivity was determined by in vitro growth in the presence of anti-malarial drugs. Parasites were genotyped using whole genome sequencing techniques. Associations between 6250 single nucleotide polymorphisms (SNPs) and resistance to individual anti-malarial agents were determined, with false discovery rate adjustment for multiple hypothesis testing. We identified expected associations in the pfcrt region with chloroquine (CQ) activity, and other novel loci associated with amodiaquine, quinazoline, and quinine activities. Signals for CQ and primaquine (PQ) overlap in and around pfcrt, and interestingly the phenotypes are inversely related for these two drugs. We catalog the variation in dhfr, dhps, mdr1, nhe, and crt, including novel SNPs, and confirm the presence of a dhfr-164L quadruple mutant in coastal Kenya. Mutations implicated in sulfadoxine-pyrimethamine resistance are at or near fixation in this sample set.

Conclusions/Significance

Sequence-based GWA studies are powerful tools for phenotypic association tests. Using this approach on falciparum parasites from coastal Kenya we identified known and previously unreported genes associated with phenotypic resistance to anti-malarial drugs, and observe in high-resolution haplotype visualizations a possible signature of an inverse selective relationship between CQ and PQ.     

Commitment to gametocytogenesis in Plasmodium falciparum

To achieve transmission, a subpopulation of asexually dividing bloodstream forms of the human malaria parasite Plasmodium falciparum withdraws from the cell cycle to develop into gametocytes - cells specialized for sexual reproduction and invasion of the mosquito vector. For natural selection to maximize transmission to new hosts, a balance must have evolved between asexual replication and sexual differentiation. Here, Mike Dyer and Karen Day consider observations on the process of commitment to gametocytogenesis and use this information as the framework for a model that begins to explain the control of the dynamics between asexual and sexual development.     

Susceptibility of Colombian Plasmodium falciparum isolates to 4-aminoquinolines and the definition of amodiaquine resistance in vitro

There are wide variations in the threshold used to define in vitro resistance of Plasmodium falciparum to amodiaquine (AQ), probably due to differences in methodology and interpretation. In vitro susceptibility data of Colombian P. falciparum strains to AQ and N-desethylamodiaquine is used to illustrate the need to standardized methodologies and compare inhibitory concentrations, instead of resistant/susceptible phenotypes, when studying the mechanisms of resistance to AQ and monitoring drug susceptibility trends in the field.     

Continuous Cultivation and Drug Susceptibility Testing of Plasmodium falciparum in a Malaria Endemic Area

ABSTRACT Isolates (UCH-23 and OM) and cloned strains of Plasmodium falciparum (Clones W-2 and D-6) were maintained in continuous culture for 28 to 150 days using culture media supplemented with 10% (v/v) heat inactivated semi-immune human plasma. Microscopic appearance and growth rates (R) of the parasites in media supplemented with semi-immune human plasma [R = 1.13 (W-2), 0.92 (D-6), 0.75 (OM) and 0.84 (UCH-23)] were comparable to those of parallel cultures maintained in media supplemented with 10% (v/v) heat inactivated non-immune human plasma [R = 1.42 (W-2), 0.83 (D-6), 0.66 (OM) and 0.89 (UCH-23)]. In addition, IC50 for chloroquine and mefloquine against the two cloned strains of P. falciparum maintained in culture media supplemented with either non-immune human plasma or semi-immune human plasma were identical. Although growth rates of new isolates (UCH-23 and OM) fluctuated over time, they stabilized between the 12th and 19th day of adaptation to culture. This fluctuation in growth rates of the new isolates underscores the influence of population dynamics during adaptation of P. falciparum to continuous culture. Sixty-eight percent of the primary isolates (170 of 250) obtained from patients in Ibadan were successfully adapted and maintained in continuous culture using semi-immune human plasma. The results of these studies indicate that semi-immune human plasma is a suitable supplement for continuous cultivation and drug susceptibility testing of P. falciparum. This finding will have practical implications in malaria endemic areas where difficulties in obtaining non-immune human plasma or serum limits establishment of continuous culture of P. falciparum and its application in studies on malaria.     

Plasmodium falciparum: selenium-induced cytotoxicity to P. falciparum

The in vitro antimalarial activity of sodium selenite (NaSe) was investigated and the mechanism of its action was studied. NaSe had antimalarial activity against both the chloroquine-susceptible strain FCR-3 and chloroquine-resistant strain K-1 of Plasmodium falciparum. The shrunken cytoplasm of the parasite was observed in a smear 12 h after treatment with NaSe. Co-treatment with copper sulfate (CuSO(4)) in culture did not affect the antimalarial activity of NaSe, but NaSe cytotoxicity against the mammalian cell line Alexander was decreased significantly. The intracellular reduced glutathione level of parasitized red blood cells was decreased significantly by treatment with NaSe, and the decrease was consistent with their mortality. Treatment with NaSe had a strong inhibitory effect on plasmodial development, and NaSe cytotoxicity to human cells was decreased by co-treatment with CuSO(4). These results suggest that co-treatment with NaSe and CuSO(4) may be useful as a new antimalarial therapy.     

Drug Resistance and in Vitro Susceptibility of Plasmodium falciparum in Thailand during 1988-2003

The aim of the present study was to investigate antimalarial drug pressure resulting from the clinical use of different antimalarials in Thailand. The phenotypic diversity of the susceptibility profiles of antimalarials, i.e., chloroquine (CQ), quinine (QN), mefloquine (MQ), and artesunate (ARS) in Plasmodium falciparum isolates collected during the period from 1988 to 2003 were studied. P. falciparum isolates from infected patients were collected from the Thai-Cambodian border area at different time periods (1988-1989, 1991-1992, and 2003), during which 3 different patterns of drug use had been implemented: MQ + sulphadoxine (S) + pyrimethamine (P), MQ alone and MQ + ARS, respectively. The in vitro drug susceptibilities were investigated using a method based on the incorporation of [³H] hypoxanthine. A total of 50 isolates were tested for susceptibilities to CQ, QN, MQ, and ARS. Of these isolates, 19, 16, and 15 were adapted during the periods 1988-1989, 1991-1993, and 2003, respectively. P. falciparum isolates collected during the 3 periods were resistant to CQ. Sensitivities to MQ declined from 1988 to 2003. In contrast, the parasite was sensitive to QN, and similar sensitivity profile patterns were observed during the 3 time periods. There was a significantly positive but weak correlation between the IC₅₀ values of CQ and QN, as well as between the IC₅₀ values of QN and MQ. Drug pressure has impact on sensitivity of P. falciparum to MQ. A combination therapy of MQ and ARS is being applied to reduce the parasite resistance, and also increasing the efficacy of the drug.     

Susceptibility of owl monkeys to Plasmodium falciparum infection in relation to location of origin, phenotype, and karyotype.

The relationship among geographic origin, phenotype, karyotype, and susceptibility of owl monkeys to 2 strains of Plasmodium falciparum was investigated. Owl monkeys from Columbia and Panama were both susceptible to fatal infections with the Asian FVO (Vietnam-Oak Knoll) strain of P. falciparum. However, when inoculated with the African FUP (Uganda-Palo Alto) strain, most Colombian owl monkeys developed fatal or potentially fatal (bled out with parasitemias of over 25%) infections, but Panamanian monkeys generally survived. Colombian and Panamanian monkeys that spontaneously recovered from malaria infection were phenotypically indistinguishable from those which died. Karyotype analysis revealed that animals considered in this study were either Karyotype II (54 chromosomes) or II (53 chromosomes). Karyotype differences between individual monkeys did not correlate with increased susceptibility or resistance to malaria. Thus, the country of origin of owl monkeys appears to play a more important role in host susceptibility to malaria infection than karyotype.     

Naturally acquired immunity to Plasmodium falciparum

Malaria infections induce multiple humoral and cellular responses, most of which are probably not protective. This discussion of the epidemiology of acquired immunity to malaria will concentrate on two main areas: first, the relationship between parasitism and disease in endemic settings and the contraints placed on determining which responses are important in acquired protective immunity; second, the central importance of antigenic diversity in the host-parasite relationship. The emphasis throughout, unless otherwise stated, will be on the major human patbogen Plasmodium falciparum.     

Stage-dependent toxicity of N-acetyl-glucosamine to Plasmodium falciparum

The effects of N-acetyl-glucosamine on growth of synchronized cultures of Plasmodium falciparum were assessed by morphological observations and by measurement of parasite incorporation of 3H-hypoxanthine. Inhibition of 3H-hypoxanthine incorporation was more marked during the later stages of the erythrocytic cycle. At concentrations of the sugar below 20 mM, however, the deleterious effects were mainly a result of failure of released merozoites to invade erythrocytes, rather than a failure of schizonts to mature or release merozoites. These results are compatible with the hypothesis that a lectin-like substance on the merozoite interacts with a surface glycoprotein on the red cell and that sugar residues on this glycoprotein may be involved in this recognition.     

Stage-specific sensitivity of Plasmodium falciparum to antifolates

Highly synchronous cultures of Plasmodium falciparum were exposed to therapeutic concentrations of sulfadoxine or pyrimethamine at different developmental stages to investigate the effect on subsequent growth. Morphological observations showed that schizont formation from uninuclear trophozoites was the only process inhibited by the drugs. Segmentation of mature schizonts, merozoite invasion and development of the ring stage remained unaffected. These results support earlier reports suggesting that DNA synthesis is most pronounced in 32-42 h old trophozoites. The possible relevance of our results to the metabolism of P. falciparum is discussed.     

Glycobiology of Plasmodium falciparum

The human malaria parasite, Plasmodium falciparum, has as its only glycoconjugate GPI anchors. These structures, present in essentially all parasite surface proteins, are associated with disease pathology. In contrast, the parasite depends for essential recognition events on saccharides associated with host cell glycoproteins and proteoglycans.