The evolution and diversity of a low complexity vaccine candidate,merozoite surface protein 9 (MSP-9), in Plasmodium vivax and closely related species

The merozoite surface protein-9 (MSP-9) has been considered a target for an anti-malarial vaccine since it is one of many proteins involved in the erythrocyte invasion, a critical step in the parasite life cycle. Orthologs encoding this antigen have been found in all known species of Plasmodium parasitic to primates. In order to characterize and investigate the extent and maintenance of MSP-9 genetic diversity, we analyzed DNA sequences of the following malaria parasite species: Plasmodium falciparum, Plasmodium reichenowi, Plasmodium chabaudi, Plasmodium yoelii, Plasmodium berghei, Plasmodium coatneyi, Plasmodium gonderi, Plasmodium knowlesi, Plasmodium inui, Plasmodium simiovale, Plasmodium fieldi, Plasmodium cynomolgi and Plasmodium vivax and evaluated the signature of natural selection in all MSP-9 orthologs. Our findings suggest that the gene encoding MSP-9 is under purifying selection in P. vivax and closely related species. We further explored how selection affected different regions of MSP-9 by comparing the polymorphisms in P. vivax and P. falciparum, and found contrasting patterns between these two species that suggest differences in functional constraints. This observation implies that the MSP-9 orthologs in human parasites may interact differently with the host immune response. Thus, studies carried out in one species cannot be directly translated into the other.     

Diagnosis of Plasmodium vivax malaria using a specific deoxyribonucleic acid probe

A deoxyribonucleic acid (DNA) probe which specifically distinguishes Plasmodium vivax from P. falciparum malaria has been derived from a P. vivax genomic DNA library. This probe, VPL101, consists of 3.2 kilobase pairs and does not hybridize with up to 6 micrograms of human or P. falciparum DNA. VPL101 contains at least two copies of a 205 base pair repeat sequence. The subcloned repeat probe, VPL101/5, reacted with 73 of 76 microscopically diagnosed P. vivax samples but not with any of 17 human DNA samples or any of 8 P. falciparum DNA samples from cultured parasites. It was possible to detect P. vivax in mixed infections in which only P. falciparum parasites were identifiable by microscopy. This P. vivax DNA probe provides a useful epidemiological tool for malaria control programmes.     

Detection of all human Plasmodium species by a telomeric DNA fragment cloned from Plasmodium berghei

A telomeric DNA fragment that was cloned from Plasmodium berghei was used to detect the genomic DNA of P. falciparum, P. vivax, P. malariae, and P. ovale. The fragment hybridized to the DNA of all four of these human Plasmodium species and can be used as an interspecific probe to detect human malaria.     

Distribution of survival times of deliberate Plasmodium falciparum infections in tertiary syphilis patients

Survival time data of Plasmodium falciparum infections from deliberate infection of human subjects with P. falciparum between 1940 and 1963 as a treatment for neurosyphilis in the USA (Georgia) have been used to test the fits of five commonly used parametric distributions for survival times using quantile-quantile plots. Our results suggest that the best fit is obtained from the Gompertz or Weibull distributions. This result has important implications for mathematical modelling of malaria, which has for the past century exclusively assumed that the duration of malaria infections has an exponential distribution. It is desirable to know the correct distribution because its shape profoundly influences the length of monitoring needed in an intervention programme for eliminating or reducing malaria.     

Susceptibility of Anopheles culicifacies species A and B to Plasmodium vivax and Plasmodium falciparum as determined by immunoradiometric assay

We have used a two-site immunoradiometric assay and species-specific antisporozoite monoclonal antibodies to determine the relative roles that sibling species A and B of the Anopheles culicifacies complex play in malaria transmission in western Uttar Pradesh, India. The results unequivocally establish species A as the primary vector of both Plasmodium vivax and P. falciparum in this area. Our results indicate active transmission of P. vivax from May to October and of P. falciparum from August to December. The identification of species A as the primary malaria vector in northern India will now allow suitable malaria control strategies to be designed.     

Haematinic treatment of anaemia increases the risk of Plasmodium vivax malaria in pregnancy

Nutritional deficiency and malaria are 2 major causes of anaemia during pregnancy in tropical areas. The relationship between anaemia, its treatment with iron and folate, and malaria was studied in a prospective cohort of 2112 pregnant Karen women on the north-western border of Thailand between 1993 and 1997. The development of Plasmodium vivax malaria was associated with a past mean haematocrit > 30% (hazard ratio = 1.5, 95% CI 1.2-2, P = 0.001) and recent (< or = 30 d) iron and folate supplementation (hazard ratio = 1.7, 95% CI 1.1-2.6, P = 0.01). There were no associations with P. falciparum infections. Plasmodium vivax has a predilection for young erythrocytes, and these results suggest that pregnant women with larger numbers of circulating young red cells are at greater risk of developing P. vivax malaria. In P. vivax-endemic areas, systematic iron and folate supplementation confers both benefit and risk in pregnancy.     

Concurrent dengue and malaria due to Plasmodium falciparum and P. vivax

Concurrent infections of dengue and malaria are rare. We report a case of dengue fever with acute malaria due to Plasmodium falciparum and P. vivax in which the presence of mixed infection with P. vivax was overlooked and confirmed later on during recurrence of the fever that had initially responded to conventional antimalarial treatment and symptomatic treatment for dengue fever. We suggest that in concurrent infections of dengue and malaria, possibility of mixed infection with various Plasmodium species should be excluded to ensure a better treatment outcome.     

Pseudomonas aeruginosa septicaemia in a patient with severe Plasmodium falciparum

This report describes a Danish patient with severe Plasmodium falciparum infection and Pseudomonas aeruginosa septicaemia. The patient had been sailing along the coast of West Africa for ten years without taking any antimalaria prophylaxis and without any apparent previous history of malaria. He presented with severe form of malaria, progressing rapidly into coma and died within a short time. P. aeruginosa was isolated from his blood taken on the day of admission. His neutrophils were all occupied by P. falciparum. The unusual combination of severe falciparum malaria infection and P. aeruginosa septicaemia with extensive involvement of neutrophils lends further support for the role of phagocytic defence in malaria.     

Plasmodium vivax malaria

We report 11 cases of severe Plasmodium vivax malaria in Bikaner (western India). Patients exhibited cerebral malaria, renal failure, circulatory collapse, severe anemia, hemoglobinurea, abnormal bleeding, acute respiratory distress syndrome, and jaundice. Peripheral blood microscopy, parasite antigen-based assays, and parasite 18s rRNA gene-based polymerase chain reaction showed the presence of P. vivax and absence of P. falciparum.     

Plasmodium malariae in Haitian refugees, Jamaica

Since 1963, reported malaria transmission in Haiti has been restricted to Plasmodium falciparum. However, screening of Haitian refugees in Jamaica in 2004, by microscopic examination, identified P. falciparum, P. vivax, and P. malariae. PCR confirmed the P. malariae and P. falciparum but not P. vivax infections. DNA sequencing and rRNA gene sequences showed transmission of P. malariae. This report confirms that P. malariae is still being transmitted in Haiti.     

Acute-phase proteins in pregnant Sudanese women with severe Plasmodium falciparum malaria

A case-control study was carried out in Kassala and Medani Maternity Hospitals in Sudan to investigate acute-phase proteins [haptoglobin, C-reactive protein (CRP), ferritin and albumin] in three groups of pregnant women (32 in each arm) comprising those with severe Plasmodium falciparum malaria or uncomplicated P. falciparum malaria and healthy controls. Whilst there was no significant difference in the levels of albumin and haptoglobin, ferritin and CRP levels were significantly higher in pregnant women with severe P. falciparum malaria. There were significant positive correlations between parasite count and haptoglobin, and medium positive correlations between parasite count and CRP.     

Geographic differentiation of polymorphism in the Plasmodium falciparum malaria vaccine candidate gene SERA5

SERA5 is regarded as a promising malaria vaccine candidate of the most virulent human malaria parasite Plasmodium falciparum. SERA5 is a 120 kDa abundantly expressed blood-stage protein containing a papain-like protease. Since substantial polymorphism in blood-stage vaccine candidates may potentially limit their efficacy, it is imperative to fully investigate polymorphism of the SERA5 gene (sera5). In this study, we performed evolutionary and population genetic analysis of sera5. The level of inter-species divergence (kS=0.076) between P. falciparum and Plasmodium reichenowi, a closely related chimpanzee malaria parasite is comparable to that of housekeeping protein genes. A signature of purifying selection was detected in the proenzyme and enzyme domains. Analysis of 445 near full-length P. falciparum sera5 sequences from nine countries in Africa, Southeast Asia, Oceania and South America revealed extensive variations in the number of octamer repeat (OR) and serine repeat (SR) regions as well as substantial level of single nucleotide polymorphism (SNP) in non-repeat regions (2562 bp). Remarkably, a 14 amino acid sequence of SERA5 (amino acids 59-72) that is known to be the in vitro target of parasite growth inhibitory antibodies was found to be perfectly conserved in all 445 worldwide isolates of P. falciparum evaluated. Unlike other major vaccine target antigen genes such as merozoite surface protein-1, apical membrane antigen-1 or circumsporozoite protein, no strong evidence for positive selection was detected for SNPs in the non-repeat regions of sera5. A biased geographical distribution was observed in SNPs as well as in the haplotypes of the sera5 OR and SR regions. In Africa, OR- and SR-haplotypes with low frequency (<5%) and SNPs with minor allele frequency (<5%) were abundant and were mostly continent-specific. Consistently, significant genetic differentiation, assessed by the Wright's fixation index (Fst) of inter-population variance in allele frequencies, was detected for SNPs and both OR- and SR-haplotypes among almost all parasite populations. The exception was parasite populations between Tanzania and Ghana, suggesting frequent gene flow in Africa. The present study points to the importance of investigating whether biased geographical distribution for SNPs and repeat variants in the OR and SR regions affect the reactivity of human serum antibodies to variants.     

Severe imported Plasmodium falciparum malaria, France, 1996-2003

Little is known about severe imported Plasmodium falciparum malaria in industrialized countries where the disease is not endemic because most studies have been case reports or have included <200 patients. To identify factors independently associated with the severity of P. falciparum, we conducted a retrospective study using surveillance data obtained from 21,888 P. falciparum patients in France during 1996-2003; 832 were classified as having severe malaria. The global case-fatality rate was 0.4% and the rate of severe malaria was ≈3.8%. Factors independently associated with severe imported P. falciparum malaria were older age, European origin, travel to eastern Africa, absence of chemoprophylaxis, initial visit to a general practitioner, time to diagnosis of 4 to 12 days, and diagnosis during the fall-winter season. Pretravel advice should take into account these factors and promote the use of antimalarial chemoprophylaxis for every traveler, with a particular focus on nonimmune travelers and elderly persons.     

Folate metabolism pathway and Plasmodium falciparum malaria infection in pregnancy

Malaria induced by Plasmodium falciparum is a major cause of mortality. P. falciparum has the ability to use host plasma folate as its primary folate source. Folate is a cofactor needed for both malaria parasite growth and host erythrocyte production. This review examines the possible impairment of the folate-mediated one-carbon metabolism pathway as a result of P. falciparum malaria infection during pregnancy. Folate deficiency during malaria infection is presented, with an emphasis on the controversy regarding the decrease of plasma or erythrocyte folate secondary to malaria. Maternal folate deficiency increases the risk of adverse pregnancy outcomes. Functional folate deficiency and/or increased plasma homocysteine levels during pregnancy of infected women in areas endemic for malaria is a probable scenario accentuating the impairment of placenta function leading to the occurrence of neural tube defects, low birth weights, and intrauterine growth retardations. Potential questions that may be answered in future investigations using an appropriate protocol to study pregnant women with malaria are also addressed.     

Effects of untreated bed nets on the transmission of Plasmodium falciparum, P. vivax and Wuchereria bancrofti in Papua New Guinea

The impact of untreated bed nets on the transmission of human malaria and filariasis in a village in a hyperendemic area of Papua New Guinea was studied. In anopheline mosquitoes, the Plasmodium falciparum sporozoite antigen positivity rate, filarial infection rates and human blood indices dropped significantly after bed nets were introduced. This reduction in human-vector contact did not affect mosquito density as no significant difference in either landing rates or indoor resting catches was found. The number of bed nets in a house and ownership of dogs were factors significantly associated with a reduction in the number of indoor resting mosquitoes. However, the reduction in the P. falciparum sporozoite antigen rate in mosquitoes was not accompanied by a reduction in either malaria parasite or antibody prevalences or titres against the P. falciparum circumsporozoite protein.     

The clinical and parasitological presentation of Plasmodium falciparum malaria in Uganda is unaffected by HIV-1 infection

The relation between Plasmodium falciparum malaria and symptomatic human immunodeficiency virus 1 (HIV-1) infection was investigated in paediatric and adult patients in Kampala, Uganda, from 1987 to 1989. Both infections contributed largely to hospital morbidity. Of 1527 clinically suspicious in-patients, 61% were positive for HIV-1 infection. 52% of patients with positive HIV-1 serology fulfilled the World Health Organization clinical case definition for acquired immune deficiency syndrome (AIDS) in Africa. No association could be found between HIV-1 infection and malaria either in paediatrics or in adults. P. falciparum parasitaemia was present in 18% of all patients and no differences in prevalence of malaria infection or in parasite density could be demonstrated between HIV-1 positive and HIV-1 negative patients. The comparison of clinical symptoms showed typical differences in AIDS-related morbidity but no difference in malaria-specific morbidity. Also, the response to malaria treatment was the same in HIV-1 positive and HIV-1 negative patients. P. falciparum malaria does not appear to act as an opportunistic agent in AIDS patients in Uganda.     

Infectivity of Plasmodium falciparum gametocytes to Anopheles arabiensis after treatment with sulfadoxine-pyrimethamine

Sulfadoxine-pyrimethamine induces increased gametocytaemia when used for treating Plasmodium falciparum malaria. Laboratory-reared Anopheles arabiensis mosquitoes were fed with blood from patients with post-therapeutic gametocytaemia using a membrane feeder. Fourteen days later the heads and thoraxes of 613 mosquitoes were negative for P. falciparum sporozoites by enzyme-linked immunosorbent assay.     

Assessing the adequacy of attenuation of genetically modified malaria parasite vaccine candidates

The critical first step in the clinical development of a malaria vaccine, based on live-attenuated Plasmodium falciparum sporozoites, is the guarantee of complete arrest in the liver. We report on an approach for assessing adequacy of attenuation of genetically attenuated sporozoites in vivo using the Plasmodium berghei model of malaria and P. falciparum sporozoites cultured in primary human hepatocytes. We show that two genetically attenuated sporozoite vaccine candidates, Δp52+p36 and Δfabb/f, are not adequately attenuated. Sporozoites infection of mice with both P. berghei candidates can result in blood infections. We also provide evidence that P. falciparum sporozoites of the leading vaccine candidate that is similarly attenuated through the deletion of the genes encoding the proteins P52 and P36, can develop into replicating liver stages. Therefore, we propose a minimal set of screening criteria to assess adequacy of sporozoite attenuation necessary before advancing into further clinical development and studies in humans.