Visceral Leishmaniasis in Traveler to Guyana Caused by Leishmania siamensis, London, UK.

The parasite Leishmania siamensis is a zoonotic agent of leishmaniasis; infection in animals has been documented in Europe and the United States. Reported authochthonous human infections have been limited to Thailand. We report a case of human visceral Leishmania siamensis infection acquired in Guyana, suggesting colonization in South America.

The design and evaluation of a shaped filter collection device to sample and store defined volume dried blood spots from finger pricks.

BACKGROUND: Dried blood spots are a common medium for collecting patient blood prior to testing for malaria by molecular methods. A new shaped filter device for the quick and simple collection of a designated volume of patient blood has been designed and tested against conventional blood spots for accuracy and precision. METHODS: Shaped filter devices were laser cut from Whatman GB003 paper to absorb a 20 µl blood volume. These devices were used to sample Plasmodium falciparum infected blood and the volume absorbed was measured volumetrically. Conventional blood spots were made by pipetting 20 µl of the same blood onto Whatman 3MM paper. DNA was extracted from both types of dried blood spot using Qiagen DNA blood mini or Chelex extraction for real-time PCR analysis, and PURE extraction for malaria LAMP testing. RESULTS: The shaped filter devices collected a mean volume of 21.1 µl of blood, with a coefficient of variance of 8.1%. When used for DNA extraction by Chelex and Qiagen methodologies the mean number of international standard units of P. falciparum DNA recovered per µl of the eluate was 53.1 (95% CI: 49.4 to 56.7) and 32.7 (95% CI: 28.8 to 36.6), respectively for the shaped filter device, and 54.6 (95% CI: 52.1 to 57.1) and 12.0 (95% CI: 9.9 to 14.1), respectively for the 3MM blood spots. Qiagen extraction of 200 µl of whole infected blood yielded 853.6 international standard units of P. falciparum DNA per µl of eluate. CONCLUSIONS: A shaped filter device provides a simple way to quickly sample and store a defined volume of blood without the need for any additional measuring devices. Resultant dried blood spots may be employed for DNA extraction using a variety of technologies for nucleic acid amplification without the need for repeated cleaning of scissors or punches to prevent cross contamination of samples and results are comparable to traditional DBS.

A lab-on-chip for malaria diagnosis and surveillance.

BACKGROUND: Access to timely and accurate diagnostic tests has a significant impact in the management of diseases of global concern such as malaria. While molecular diagnostics satisfy this need effectively in developed countries, barriers in technology, reagent storage, cost and expertise have hampered the introduction of these methods in developing countries. In this study a simple, lab-on-chip PCR diagnostic was created for malaria that overcomes these challenges. METHODS: The platform consists of a disposable plastic chip and a low-cost, portable, real-time PCR machine. The chip contains a desiccated hydrogel with reagents needed for Plasmodium specific PCR. Chips can be stored at room temperature and used on demand by rehydrating the gel with unprocessed blood, avoiding the need for sample preparation. These chips were run on a custom-built instrument containing a Peltier element for thermal cycling and a laser/camera setup for amplicon detection. RESULTS: This diagnostic was capable of detecting all Plasmodium species with a limit of detection for Plasmodium falciparum of 2 parasites/µL of blood. This exceeds the sensitivity of microscopy, the current standard for diagnosis in the field, by ten to fifty-fold. In a blind panel of 188 patient samples from a hyper-endemic region of malaria transmission in Uganda, the diagnostic had high sensitivity (97.4%) and specificity (93.8%) versus conventional real-time PCR. The test also distinguished the two most prevalent malaria species in mixed infections, P. falciparum and Plasmodium vivax. A second blind panel of 38 patient samples was tested on a streamlined instrument with LED-based excitation, achieving a sensitivity of 96.7% and a specificity of 100%. CONCLUSIONS: These results describe the development of a lab-on-chip PCR diagnostic from initial concept to ready-for-manufacture design. This platform will be useful in front-line malaria diagnosis, elimination programmes, and clinical trials. Furthermore, test chips can be adapted to detect other pathogens for a differential diagnosis in the field. The flexibility, reliability, and robustness of this technology hold much promise for its use as a novel molecular diagnostic platform in developing countries.

Clinical evaluation of a loop-mediated amplification kit for diagnosis of imported malaria.

BACKGROUND: Diagnosis of malaria relies on parasite detection by microscopy or antigen detection; both fail to detect low-density infections. New tests providing rapid, sensitive diagnosis with minimal need for training would enhance both malaria diagnosis and malaria control activities. We determined the diagnostic accuracy of a new loop-mediated amplification (LAMP) kit in febrile returned travelers. METHODS: The kit was evaluated in sequential blood samples from returned travelers sent for pathogen testing to a specialist parasitology laboratory. Microscopy was performed, and then malaria LAMP was performed using Plasmodium genus and Plasmodium falciparum-specific tests in parallel. Nested polymerase chain reaction (PCR) was performed on all samples as the reference standard. Primary outcome measures for diagnostic accuracy were sensitivity and specificity of LAMP results, compared with those of nested PCR. RESULTS: A total of 705 samples were tested in the primary analysis. Sensitivity and specificity were 98.4% and 98.1%, respectively, for the LAMP P. falciparum primers and 97.0% and 99.2%, respectively, for the Plasmodium genus primers. Post hoc repeat PCR analysis of all 15 tests with discrepant results resolved 4 results in favor of LAMP, suggesting that the primary analysis had underestimated diagnostic accuracy. CONCLUSIONS: Malaria LAMP had a diagnostic accuracy similar to that of nested PCR, with a greatly reduced time to result, and was superior to expert microscopy.

Highly sensitive detection of malaria parasitemia in a malaria-endemic setting: performance of a new loop-mediated isothermal amplification kit in a remote clinic in Uganda.

BACKGROUND: Current malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too complex for field deployment. A new commercial molecular assay based on loop-mediated isothermal amplification (LAMP) was assessed for field use. METHODS: Malaria LAMP (Eiken Chemical, Japan) was evaluated for samples from 272 outpatients at a rural Ugandan clinic and compared with expert microscopy, nested PCR, and quantitative PCR (qPCR). Two technicians performed the assay after 3 days of training, using 2 alternative blood sample-preparation methods and visual interpretation of results by fluorescence assay. RESULTS: Compared with 3-well nested PCR, the sensitivity of both LAMP and single-well nested PCR was 90%; the microscopy sensitivity was 51%. For samples with a Plasmodium falciparum qPCR titer of ≥ 2 parasites/µL, LAMP sensitivity was 97.8% (95% confidence interval, 93.7%-99.5%). Most false-negative LAMP results involved samples with parasitemia levels detectable by 3-well nested PCR but very low or undetectable by qPCR. CONCLUSIONS: Malaria LAMP in a remote Ugandan clinic achieved sensitivity similar to that of single-well nested PCR in a United Kingdom reference laboratory. LAMP dramatically lowers the detection threshold achievable in malaria-endemic settings, providing a new tool for diagnosis, surveillance, and screening in elimination strategies.

Increased sensitivity for detecting malaria parasites in human umbilical cord blood using scaled-up DNA preparation.

BACKGROUND: All mothers donating umbilical cord blood units to the NHS cord blood bank undergo an assessment for the likelihood of prior exposure to malaria infection. Those deemed at risk due to a history of travel to, or residence in, malaria endemic regions are screened serologically to detect anti-malaria antibodies. A positive result excludes the use of the cord blood for transplant therapy unless a risk assessment can ensure that malaria transmission is extremely unlikely. This paper details the screening of cord blood units from malaria serology positive mothers to detect malaria parasite DNA using a highly sensitive nested PCR. METHODS: Uninfected blood from a healthy volunteer was spiked with known quantities of malaria parasites and 5 millilitre and 200 microlitre aliquots were subjected to DNA extraction using QIAamp DNA maxi and DNA mini kits respectively. Nested PCR, to detect malarial SSU rRNA sequences, was performed on the purified DNA samples to determine the limit of detection for this assay with both extraction methodologies. Following assay validation, 54 cord blood units donated by mothers who were positive for anti-malaria antibodies were screened by this approach. RESULTS: When DNA was purified from 5 millilitres of blood it was possible to routinely detect as few as 50 malaria parasites per millilitre using nested PCR. This equates to a significant increase in the sensitivity of the current gold standard nucleic acid amplification technique used to detect malaria parasites (routinely performed from > 200 microlitre volumes of blood). None of the 54 donated cord blood units from serology positive mothers tested positive for malaria parasites using this scaled up DNA preparation method. CONCLUSION: Serological testing for malaria parasites may be overly conservative, leading to unnecessary rejection of cord blood donations that lack malaria parasites and which are, therefore, safe for use in stem cell therapy.

Gametogenesis in malaria parasites is mediated by the cGMP-dependent protein kinase.

Malaria parasite transmission requires differentiation of male and female gametocytes into gametes within a mosquito following a blood meal. A mosquito-derived molecule, xanthurenic acid (XA), can trigger gametogenesis, but the signalling events controlling this process in the human malaria parasite Plasmodium falciparum remain unknown. A role for cGMP was revealed by our observation that zaprinast (an inhibitor of phosphodiesterases that hydrolyse cGMP) stimulates gametogenesis in the absence of XA. Using cGMP-dependent protein kinase (PKG) inhibitors in conjunction with transgenic parasites expressing an inhibitor-insensitive mutant PKG enzyme, we demonstrate that PKG is essential for XA- and zaprinast-induced gametogenesis. Furthermore, we show that intracellular calcium (Ca2+) is required for differentiation and acts downstream of or in parallel with PKG activation. This work defines a key role for PKG in gametogenesis, elucidates the hierarchy of signalling events governing this process in P. falciparum, and demonstrates the feasibility of selective inhibition of a crucial regulator of the malaria parasite life cycle.

Two nonrecombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally.

BACKGROUND: Malaria in humans is caused by apicomplexan parasites belonging to 5 species of the genus Plasmodium. Infections with Plasmodium ovale are widely distributed but rarely investigated, and the resulting burden of disease is not known. Dimorphism in defined genes has led to P. ovale parasites being divided into classic and variant types. We hypothesized that these dimorphs represent distinct parasite species. METHODS: Multilocus sequence analysis of 6 genetic characters was carried out among 55 isolates from 12 African and 3 Asia-Pacific countries. RESULTS: Each genetic character displayed complete dimorphism and segregated perfectly between the 2 types. Both types were identified in samples from Ghana, Nigeria, São Tomé, Sierra Leone, and Uganda and have been described previously in Myanmar. Splitting of the 2 lineages is estimated to have occurred between 1.0 and 3.5 million years ago in hominid hosts. CONCLUSIONS: We propose that P. ovale comprises 2 nonrecombining species that are sympatric in Africa and Asia. We speculate on possible scenarios that could have led to this speciation. Furthermore, the relatively high frequency of imported cases of symptomatic P. ovale infection in the United Kingdom suggests that the morbidity caused by ovale malaria has been underestimated.

Structure and non-essential function of glycerol kinase in Plasmodium falciparum blood stages.

Malaria pathology is caused by multiplication of asexual parasites within erythrocytes, whereas mosquito transmission of malaria is mediated by sexual precursor cells (gametocytes). Microarray analysis identified glycerol kinase (GK) as the second most highly upregulated gene in Plasmodium falciparum gametocytes with no expression detectable in asexual blood stage parasites. Phosphorylation of glycerol by GK is the rate-limiting step in glycerol utilization. Deletion of this gene from P. falciparum had no effect on asexual parasite growth, but surprisingly also had no effect on gametocyte development or exflagellation, suggesting that these life cycle stages do not utilize host-derived glycerol as a carbon source. Kinetic studies of purified PfGK showed that the enzyme is not regulated by fructose 1,6 bisphosphate. The high-resolution crystal structure of P. falciparum GK, the first of a eukaryotic GK, reveals two domains embracing a capacious ligand-binding groove. In the complexes of PfGK with glycerol and ADP, we observed closed and open forms of the active site respectively. The 27 degree domain opening is larger than in orthologous systems and exposes an extensive surface with potential for exploitation in selective inhibitor design should the enzyme prove to be essential in vivo either in the human or in the mosquito.

Mitochondrial DNA targets increase sensitivity of malaria detection using loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) of DNA offers the ability to detect very small quantities of pathogen DNA following minimal tissue sample processing and is thus an attractive methodology for point-of-care diagnostics. Previous attempts to diagnose malaria by the use of blood samples and LAMP have targeted the parasite small-subunit rRNA gene, with a resultant sensitivity for Plasmodium falciparum of around 100 parasites per microl. Here we describe the use of mitochondrial targets for LAMP-based detection of any Plasmodium genus parasite and of P. falciparum specifically. These new targets allow routine amplification from samples containing as few as five parasites per microl of blood. Amplification is complete within 30 to 40 min and is assessed by real-time turbidimetry, thereby offering rapid diagnosis with greater sensitivity than is achieved by the most skilled microscopist or antigen detection using lateral flow immunoassays.

Measuring the efficacy of anti-malarial drugs in vivo: quantitative PCR measurement of parasite clearance.

BACKGROUND: Artemisinin-based combination therapy, currently considered the therapy of choice for uncomplicated Plasmodium falciparum malaria in endemic countries, may be under threat from newly emerging parasite resistance to the artemisinin family of drugs. Studies in Southeast Asia suggest some patients exhibit an extended parasite clearance time in the three days immediately following treatment with artesunate monotherapy. This phenotype is likely to become a more important trial endpoint in studies of anti-malarial drug efficacy, but currently requires frequent, closely spaced blood sampling in hospitalized study participants, followed by quantitation of parasite density by microscopy. METHODS: A simple duplex quantitative PCR method was developed in which distinct fluorescent signals are generated from the human and parasite DNA components in each blood sample. The human amplification target in this assay is the ß tubulin gene, and the parasite target is the unique methionine tRNA gene (pgmet), which exhibits perfect sequence identity in all six Plasmodium species that naturally infect humans. In a small series of malaria cases treated as hospital in-patients, the abundance of pgmet DNA was estimated relative to the human DNA target in daily peripheral blood samples, and parasite clearance times calculated. RESULTS: The qPCR assay was reproducibly able to replicate parasite density estimates derived from microscopy, but provided additional data by quantification of parasite density 24 hours after the last positive blood film. Robust estimates of parasite clearance times were produced for a series of patients with clinical malaria. CONCLUSIONS: Large studies, particularly in Africa where children represent a major proportion of treated cases, will require a simpler blood sample collection regime, and a method capable of high throughput. The duplex qPCR method tested may fulfil these criteria, and should now be evaluated in such field studies.

Novel pfdhps haplotypes among imported cases of Plasmodium falciparum malaria in the United Kingdom.

Treatment of acute malaria caused by Plasmodium falciparum may include long-half-life drugs, such as the antifolate combination sulfadoxine-pyrimethamine (SP), to provide posttreatment chemoprophylaxis against parasite recrudescence or delayed emergence from the liver. An unusual case of P. falciparum recrudescence in a returned British traveler who received such a regimen, as well as a series of 44 parasite isolates from the same hospital, was analyzed by PCR and direct DNA sequencing for the presence of markers of parasite resistance to chloroquine and antifolates. The index patient harbored a mixture of wild-type and resistant pfdhfr and pfdhps alleles upon initial presentation. During his second malaria episode, he harbored only resistant parasites, with the haplotypes IRNI (codons 51, 59, 108, and 164) and SGEAA (codons 436, 437, 540, 581, and 613) at these two loci, respectively. Analysis of isolates from 44 other patients showed that the pfdhfr haplotype IRNI was common (found in 81% of cases). The SGEAA haplotype of pfdhps was uncommon (found only in eight cases of East African origin [17%]). A previously undescribed mutation, I431V, was observed for seven cases of Nigerian origin, occurring as one of two haplotypes, VAGKGS or VAGKAA. The presence of this mutation was also confirmed in isolates of Nigerian origin from the United Kingdom Malaria Reference Laboratory. The presence of the pfdhps haplotype SGEAA in P. falciparum parasites of East African origin appears to compromise the efficacy of treatment regimens that include SP as a means to prevent recrudescence. Parasites with novel pfdhps haplotypes are circulating in West Africa. The response of these parasites to chemotherapy needs to be evaluated.

Breadth and magnitude of antibody responses to multiple Plasmodium falciparum merozoite antigens are associated with protection from clinical malaria.

Individuals living in areas where malaria is endemic are repeatedly exposed to many different malaria parasite antigens. Studies on naturally acquired antibody-mediated immunity to clinical malaria have largely focused on the presence of responses to individual antigens and their associations with decreased morbidity. We hypothesized that the breadth (number of important targets to which antibodies were made) and magnitude (antibody level measured in a random serum sample) of the antibody response were important predictors of protection from clinical malaria. We analyzed naturally acquired antibodies to five leading Plasmodium falciparum merozoite-stage vaccine candidate antigens, and schizont extract, in Kenyan children monitored for uncomplicated malaria for 6 months (n = 119). Serum antibody levels to apical membrane antigen 1 (AMA1) and merozoite surface protein antigens (MSP-1 block 2, MSP-2, and MSP-3) were inversely related to the probability of developing malaria, but levels to MSP-1(19) and erythrocyte binding antigen (EBA-175) were not. The risk of malaria was also inversely associated with increasing breadth of antibody specificities, with none of the children who simultaneously had high antibody levels to five or more antigens experiencing a clinical episode (17/119; 15%; P = 0.0006). Particular combinations of antibodies (AMA1, MSP-2, and MSP-3) were more strongly predictive of protection than others. The results were validated in a larger, separate case-control study whose end point was malaria severe enough to warrant hospital admission (n = 387). These findings suggest that under natural exposure, immunity to malaria may result from high titers antibodies to multiple antigenic targets and support the idea of testing combination blood-stage vaccines optimized to induce similar antibody profiles.

Evidence of non-neutral polymorphism in Plasmodium falciparum gamete surface protein genes Pfs47 and Pfs48/45.

Targeted disruption of particular members of the Plasmodium 6-cys protein gene family, including Ps47, Ps48/45 and Ps230, is known to dramatically affect parasite fertility. Because loci critical to fertility in many eukaryote species have been shown to be under strong positive selection, we examined sequence variation in four members of the 6-cys protein gene family in Plasmodium falciparum (Pfs36, Pfs38, Pfs47 and Pfs48/45) to determine whether genetic variation in these loci may be of functional significance. Sequence polymorphism among 11 laboratory isolates of P. falciparum was compared with divergence from the respective orthologues in the closely related species P. reichenowi, showing an almost significant skew towards within-species non-synonymous polymorphism in Pfs47 and Pfs48/45 (by the McDonald-Kreitman test) but clearly non-significant results for Pfs36 and Pfs38. A preliminary analysis of Pfs47 sequence polymorphism in field isolates of P. falciparum showed exceptionally high fixation indices (F(ST)) among geographically distinct populations, similar to results seen previously for Pfs48/45. Therefore, both Pfs47 and Pfs48/45 were further analysed by sequencing polymorphic parts of the genes from a Tanzanian population sample of oocysts (a means of analysing diploid genotypes). Both genes displayed higher inbreeding coefficients (F(IS)) compared with the average of 11 unlinked microsatellite loci. These results suggest that allelic variation in these two genes may be functionally significant in influencing mating interactions, a hypothesis that could be tested by fertilization experiments with targeted allelic replacement.

Progress and challenges towards the development of malaria vaccines.

The promise afforded by attenuated sporozoite vaccines in the 1970s led many researchers to believe that an efficacious malaria vaccine was an attainable medium-term goal. Over 30 years later, no licensed vaccine is currently available for public health intervention. This is despite global expenditure on research and development for malaria vaccines that is estimated to have increased from $US42 million in 1999 to $US84 million in 2004. Serious questions must therefore be asked: is this a good investment of research and public health funds, and are we really any nearer to producing a viable product for global use?Proponents of a malaria vaccine promote this technology as a viable way to combat both the current economic and humanitarian burden of malaria and the decreasing efficacy of many front-line antimalaria drug therapies. The recent successful phase IIb trial of the RTS,S/AS02A vaccine showed that the production of a subunit vaccine with significant efficacy is technically possible. The combined efforts and financial commitment of researchers, pharmaceutical companies, and not-for-profit organizations, including the Malaria Vaccines Initiative, have resulted in a significant scaling up in the number of products suitable for testing in humans. In addition, new technologies, such as genetically attenuated vaccines and the exploitation of malaria genomes, offer exciting possibilities for vaccine development. There is now a real possibility of producing a malaria vaccine licensed for public health. However, this positive outlook must be tempered with the challenges facing vaccine development and distribution. The efficacy levels seen with RTS,S/AS02A are well below those of all vaccines currently in use for public health. Furthermore, poor preclinical and clinical predictors of efficacy, allele-specific immunity, and an imperfect understanding of natural and induced immunity to malaria may yet delay (or even prevent) the development of a vaccine suitable for global use.

Development and clinical performance of high throughput loop-mediated isothermal amplification for detection of malaria.

BACKGROUND: Accurate and efficient detection of sub-microscopic malaria infections is crucial for enabling rapid treatment and interruption of transmission. Commercially available malaria LAMP kits have excellent diagnostic performance, though throughput is limited by the need to prepare samples individually. Here, we evaluate the clinical performance of a newly developed high throughput (HTP) sample processing system for use in conjunction with the Eiken malaria LAMP kit. METHODS: The HTP system utilised dried blood spots (DBS) and liquid whole blood (WB), with parallel sample processing of 94 samples per run. The system was evaluated using 699 samples of known infection status pre-determined by gold standard nested PCR. RESULTS: The sensitivity and specificity of WB-HTP-LAMP was 98.6% (95% CI, 95.7-100), and 99.7% (95% CI, 99.2-100); sensitivity of DBS-HTP-LAMP was 97.1% (95% CI, 93.1-100), and specificity 100% against PCR. At parasite densities greater or equal to 2 parasites/µL, WB and DBS HTP-LAMP showed 100% sensitivity and specificity against PCR. At densities less than 2 p/µL, WB-HTP-LAMP sensitivity was 88.9% (95% CI, 77.1-100) and specificity was 99.7% (95% CI, 99.2-100); sensitivity and specificity of DBS-HTP-LAMP was 77.8% (95% CI, 54.3-99.5) and 100% respectively. CONCLUSIONS: The HTP-LAMP system is a highly sensitive diagnostic test, with the potential to allow large scale population screening in malaria elimination campaigns.

Contrasting signatures of selection on the Plasmodium falciparum erythrocyte binding antigen gene family.

Erythrocyte binding antigens of Plasmodium falciparum are involved in erythrocyte invasion, and may be targets of acquired immunity. Of the five eba genes, protein products have been detected for eba-175, eba-181 and eba-140, but not for psieba-165 or ebl-1, providing opportunity for comparative analysis of genetic variation to identify selection. Region II of each of these genes was sequenced from a cross-sectional sample of parasites in an endemic Kenyan population, and the frequency distributions of polymorphisms analysed. A positive value of Tajima's D was observed for eba-175 (D=1.13) indicating an excess of intermediate frequency polymorphisms, while all other genes had negative values, the most negative being ebl-1 (D=-2.35) followed by psieba-165 (D=-1.79). The eba-175 and ebl-1 genes were then studied in a sample of parasites from Thailand, for which a positive Tajima's D value was again observed for eba-175 (D=1.79), and a negative value for ebl-1 (D=-1.85). This indicates that eba-175 is under balancing selection in each population, in strong contrast to the other members of the gene family, particularly ebl-1 and psieba-165 that may have been under recent directional selection. Population expansion simulations were performed under a neutral model, further supporting the departures from neutrality of these genes.

Orthologous gene sequences of merozoite surface protein 1 (MSP1) from Plasmodium reichenowi and P. gallinaceum confirm an ancient divergence of P. falciparum alleles.

Merozoite surface protein 1 (MSP 1) of Plasmodium falciparum has a major allelic dimorphism in the majority of its sequence, the origin and significance of which is obscure. Here, the cloning and sequencing of the msp1 gene from P. reichenowi (a chimpanzee parasite that is the nearest relative of P. falciparum) and P. gallinaceum (a malaria parasite of birds) is reported. P. reichenowi msp1 is most closely related to one allelic type (K1) of P. falciparum. The other P. falciparum major allelic type (MAD20) is very divergent from these sequences, although not as divergent as msp1 of P. gallinaceum. Assuming a date of 6 million years ago (mya) for the divergence of the P. falciparum K1 and the P. reichenowi msp1 genes (on the basis of previous estimates for these parasite species as well as host divergence times), the most recent common ancestor of the dimorphic region of msp1 would date to approximately 27mya. Thus, the P. falciparum msp1 dimorphism is confirmed as one of the oldest polymorphisms known with the exception of self-incompatibility S genes in Solanaceae. In contrast with the major allelic dimorphism, the polymorphisms present in the relatively conserved C terminus of P. falciparum msp1 appear to have arisen since the divergence of the P. falciparum and P. reichenowi msp1 genes.

Allele frequency-based analyses robustly map sequence sites under balancing selection in a malaria vaccine candidate antigen.

The Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading candidate for a malaria vaccine. Here, within-population analyses of alleles from 50 Thai P. falciparum isolates yield significant evidence for balancing selection on polymorphisms within the disulfide-bonded domains I and III of the surface accessible ectodomain of AMA1, a result very similar to that seen previously in a Nigerian population. Studying the frequency of nucleotide polymorphisms in both populations shows that the between-population component of variance (F(ST)) is significantly lower in domains I and III compared to the intervening domain II and compared to 11 unlinked microsatellite loci. A nucleotide site-by-site analysis shows that sites with exceptionally high or low F(ST) values cluster significantly into serial runs, with four runs of low values in domain I and one in domain III. These runs may map the sequences that are consistently under the strongest balancing selection from naturally acquired immune responses.

Vaccination for vivax malaria: targeting the invaders.

Among the surface-exposed antigens of the malaria parasite, those with known essential functions that can be disrupted by antibodies represent the most promising candidates for development as malaria vaccines. Two recombinant protein subunits of the Plasmodium vivax merozoite surface protein 1 have been shown to bind to reticulocytes in enzyme-linked immunosorbent assays. This article discusses the importance of such pre-clinical analyses in the validation of candidate vaccine molecules for P. vivax, given the constraints imposed by the use of primate models and the cost of producing suitable material for human trials.