Human Plasmodium vivax diversity, population structure and evolutionary origin.

More than 200 million malaria clinical cases are reported each year due to Plasmodium vivax, the most widespread Plasmodium species in the world. This species has been neglected and understudied for a long time, due to its lower mortality in comparison with Plasmodium falciparum. A renewed interest has emerged in the past decade with the discovery of antimalarial drug resistance and of severe and even fatal human cases. Nonetheless, today there are still significant gaps in our understanding of the population genetics and evolutionary history of P. vivax, particularly because of a lack of genetic data from Africa. To address these gaps, we genotyped 14 microsatellite loci in 834 samples obtained from 28 locations in 20 countries from around the world. We discuss the worldwide population genetic structure and diversity and the evolutionary origin of P. vivax in the world and its introduction into the Americas. This study demonstrates the importance of conducting genome-wide analyses of P. vivax in order to unravel its complex evolutionary history.

Multiplicity of infection and genetic diversity of Plasmodium falciparum isolates from patients with uncomplicated and severe malaria in Gezira State, Sudan.

BACKGROUND: Multiplicity and genetic diversity of Plasmodium falciparum infection might play a role in determining the clinical outcome of malaria infection and could be a fair reflection of the disease transmission rate. This study investigated the genetic diversity of P. falciparum and multiplicity of infection in relation to the severity of malaria and age of patients in Gezira State, Sudan. METHODS: A cross-sectional health facilities-based survey was conducted in Gezira State, Sudan in January 2012. A total of 140 P. falciparum malaria patients diagnosed with microscopy and confirmed using nested PCR were recruited and classified into uncomplicated malaria and severe malaria states according to the standard WHO criteria. DNA was extracted and MSP1 and MSP2 allelic families were determined using nested PCR. RESULTS: The overall multiplicity of infection (MOI) was 2.25 and 2.30 and 2.15 for uncomplicated and severe malaria respectively. There were no statistically significant differences between uncomplicated and severe malaria (SM) patient groups in MOI with regard to MSP1, MSP2 and overall MOI (Mann-Whitney U-test; all P < 0.05). The predominant MSP1 allelic families were MAD20 for uncomplicated malaria and RO33 for severe malaria. The distribution of both FC27 and IC1/3D7 MSP2 allelic families were approximately the same across disease severity. One hundred and eleven P. falciparum isolates (81 %) consisted of multiple genotypes; 71/90 (78.9 %) in uncomplicated malaria and 40/50 (85.1 %) in severe malaria patient groups. Neither MSP1 nor MSP2 allelic families showed association with malaria severity. No statistically significant differences in multiplicity of infection were observed between different age groups. CONCLUSION: In this study the majority of P. falciparum isolates from uncomplicated and severe malaria patients consisted of multiple genotypes. Further molecular epidemiological studies delineate the link between P. falciparum genotype with the malaria phenotype in different regions are encouraged.

Transmission of Plasmodium vivax in Duffy-negative individuals in central Sudan.

BACKGROUND: Due to the recently observed rise in Plasmodium vivax incidence in Sudan and reported transmission in Duffy-negative individuals; we aimed to assess the possibility of P. vivax transmission in Duffy-negative individuals in Gezira state, central Sudan. METHOD: A total of 126 suspected malaria patients were diagnosed with P. vivax infection using microscopy, RDT and PCR. PCR-RFLP was used to genotype participants Duffy status. RESULTS: Forty eight (38%) were positive for P. vivax infection by PCR. Four patients (8.3%) were homozygous Duffy-negative. CONCLUSION: These results confirm that P. vivax can infect Duffy-negative individuals, suggesting alternative mechanisms to bind and invade erythrocytes.

Impacts of agricultural practices on insecticide resistance in the malaria vector Anopheles arabiensis in Khartoum State, Sudan.

BACKGROUND: Agricultural pesticides may play a profound role in selection of resistance in field populations of mosquito vectors. The objective of this study is to investigate possible links between agricultural pesticide use and development of resistance to insecticides by the major malaria vector Anopheles arabiensis in northern Sudan. METHODOLOGY/PRINCIPAL FINDINGS: Entomological surveys were conducted during two agricultural seasons in six urban and peri-urban sites in Khartoum state. Agro-sociological data were collected from 240 farmers subjected to semi-structured questionnaires based on knowledge attitude and practice (KAP) surveys. Susceptibility status of An. arabiensis (n=6000) was assessed in all sites and during each season using WHO bioassay tests to DDT, deltamethrin, permethrin, Malathion and bendiocarb. KAP analysis revealed that pesticide application was common practice among both urban and peri-urban farmers, with organophosphates and carbamates most commonly used. Selection for resistance is likely to be greater in peri-urban sites where farmers apply pesticide more frequently and are less likely to dispose of surpluses correctly. Though variable among insecticides and seasons, broad-spectrum mortality was slightly, but significantly higher in urban than peri-urban sites and most marked for bendiocarb, to which susceptibility was lowest. Anopheles arabiensis from all sites showed evidence of resistance or suspected resistance, especially pyrethroids. However, low-moderate frequencies of the L1014F kdr allele in all sites, which was very strongly associated with DDT, permethrin and deltamethrin survivorship (OR=6.14-14.67) suggests that resistance could increase rapidly. CONCLUSIONS: Ubiquitous multiple-resistance coupled with presence of a clear mechanism for DDT and pyrethroids (kdr L1014F) in populations of An. arabiensis from Khartoum-Sudan suggests careful insecticide management is essential to prolong efficacy. Our findings are consistent with agricultural insecticide use as a source of selection for resistance and argue for coordination between the integrated vector control program and the Ministry of Agriculture to permit successful implementation of rational resistance management strategies.

Vaccination with Plasmodium knowlesi AMA1 formulated in the novel adjuvant co-vaccine HT™ protects against blood-stage challenge in rhesus macaques.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading blood stage vaccine candidate. Plasmodium knowlesi AMA1 (PkAMA1) was produced and purified using similar methodology as for clinical grade PfAMA1 yielding a pure, conformational intact protein. Combined with the adjuvant CoVaccine HT™, PkAMA1 was found to be highly immunogenic in rabbits and the efficacy of the PkAMA1 was subsequently tested in a rhesus macaque blood-stage challenge model. Six rhesus monkeys were vaccinated with PkAMA1 and a control group of 6 were vaccinated with PfAMA1. A total of 50 µg AMA1 was administered intramuscularly three times at 4 week intervals. One of six rhesus monkeys vaccinated with PkAMA1 was able to control parasitaemia, upon blood stage challenge with P. knowlesi H-strain. Four out of the remaining five showed a delay in parasite onset that correlated with functional antibody titres. In the PfAMA1 vaccinated control group, five out of six animals had to be treated with antimalarials 8 days after challenge; one animal did not become patent during the challenge period. Following a rest period, animals were boosted and challenged again. Four of the six rhesus monkeys vaccinated with PkAMA1 were able to control the parasitaemia, one had a delayed onset of parasitaemia and one animal was not protected, while all control animals required treatment. To confirm that the control of parasitaemia was AMA1-related, animals were allowed to recover, boosted and re-challenged with P. knowlesi Nuri strain. All control animals had to be treated with antimalarials by day 8, while five out of six PkAMA1 vaccinated animals were able to control parasitaemia. This study shows that: i) Yeast-expressed PkAMA1 can protect against blood stage challenge; ii) Functional antibody levels as measured by GIA correlated inversely with the day of onset and iii) GIA IC(50) values correlated with estimated in vivo growth rates.