An update on the search for a Plasmodium vivax vaccine

Although Plasmodium falciparum is the leading cause of morbidity and mortality due to malaria worldwide, nearly 2.5 billion people, mostly outside Africa, are also at risk from malaria caused by Plasmodium vivax infection. Currently, almost all efforts to develop a malaria vaccine have focused on P. falciparum. For example, there are 23 P. falciparum vaccine candidates undergoing advanced clinical studies and only two P. vivax vaccine candidates being tested in preliminary (Phase I) clinical trials, with few others being assessed in preclinical studies. More investment and a greater effort toward the development of P. vivax vaccine components for a multi-species vaccine are required. This is mainly because of the wide geographical coexistence of both parasite species but also because of increasing drug resistance, recent observations of severe and lethal P. vivax cases and relapsing parasite behaviour. Availability of the P. vivax genome has contributed to antigen discovery but new means to test vaccines in future trials remain to be designed.     

Plasmodium vivax: parasitemia determination by real-time quantitative PCR in Aotus monkeys

Plasmodium vivax and Plasmodium falciparum are the two prevalent human malaria species. A Colombian P. vivax wild strain has been adapted in Aotus nancymaae monkeys for use in further biological and immunological studies. We present data validating a real-time PCR assay quantifying P. vivax parasitemia, using the small subunit ribosomal RNA genes as an amplification target. P. vivax species-specific primers were designed on the 18S ribosomal gene V8 region, for amplifying both asexual and sporozoite ssrRNA genes. The assay detects amplification products bound to fluorescent SYBR-Green I dye using Perkin-Elmer GeneAmp-5700-SDS. Linear range standard curves from 6 DNA concentration logs (+0.99 correlation coefficients) were obtained. Standard curves were constructed using a plasmid containing target gene for real-time PCR amplification. This P. vivax specific assay is very sensitive, having a three parasite detection limit, and is reproducible and accurate. It involves a "closed-tube" PCR, avoids time-consuming post-PCR manipulation, and decreases potential PCR contamination.     

The baseline distribution of malaria in the initial phase of elimination in Sabang Municipality, Aceh Province, Indonesia

ABSTRACT: BACKGROUND: Sabang Municipality, in Aceh Province, Indonesia, plans to initiate a malaria elimination programme in 2013. A baseline survey of the distribution of malaria in the municipality was conducted to lay the foundations for an evidence-based programme and to assess the island's readiness to begin the elimination process. METHODS: The entire population of the municipality was screened for malaria infection and G6PD deficiency. Specimens collected included blood slides, blots and tubes for selected households. Results and Discussion Samples were collected from 16,229 residents. Microscopic examination of the blood smears revealed 12 malaria infections; 10 with Plasmodium falciparum and 2 with Plasmodium vivax. To confirm the parasite prevalence, polymerase chain reaction (PCR) diagnosis was performed on the entire positive cases by microscopy and randomized 10% of the microscopically negative blood samples. PCR revealed an additional 11 subjects with malaria; one P. falciparum infection from the village of Paya Keunekai, and nine P. vivax infections and one mixed P. falciparum/P. vivax infection from the village of Batee Shok. The overall slide positivity rate was 0.074% (CI 95%: 0.070 - 0.078) and PCR corrected prevalence 0,590% (CI 95%: 0.582 - 0.597). Analysis of 937 blood samples for G6PD deficiency revealed two subjects (0.2%) of deficient G6PD. Analysis of several genes of the parasite, such as Pfdhfr, Pfdhps, Pfmdr1, Pfcrt, Pfmsp1, Pfmsp2, Pvdhfr, Pvdhps, Pvmdr1 and host gene, such as G6PD gene revealed that both P. falciparum and P. vivax carried the mutation associated with chloroquine resistance. CONCLUSION: Malariometric and host genetic analysis indicated that there is a low prevalence of both malaria and G6PD deficiency in the population of Sabang Municipality. Nevertheless, malaria cases were clustered in three rural villages and efforts for malaria elimination in Sabang should be particularly focused on those three villages.     

A new single-step PCR assay for the detection of the zoonotic malaria parasite Plasmodium knowlesi

BACKGROUND: Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection. METHODOLOGY AND SIGNIFICANT FINDINGS: We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites. CONCLUSIONS: The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi.     

Identification and characterisation of the Plasmodium vivax rhoptry-associated protein 2

Plasmodium vivax is currently the most widespread of the four parasite species causing malaria in humans around the world. It causes more than 75 million clinical episodes per year, mainly on the Asian and American continents. Identifying new antigens to be further tested as anti-P. vivax vaccine candidates has been greatly hampered by the difficulty of maintaining this parasite cultured in vitro. Taking into account that one of the most promising vaccine candidates against Plasmodium falciparum is the rhoptry-associated protein 2, we have identified the P. falciparum rhoptry-associated protein 2 homologue in P. vivax in the present study. This protein has 400 residues, having an N-terminal 21 amino-acid stretch compatible with a signal peptide and, as occurs with its falciparum homologue, it lacks repeat sequences. The protein is expressed in asexual stage P. vivax parasites and polyclonal sera raised against this protein recognised a 46 kDa band in parasite lysate in a Western blot assay.     

Cloning, overexpression, purification and characterization of Plasmodium knowlesi lactate dehydrogenase

Plasmodial lactate dehydrogenase, key enzyme of anaerobic glycolysis, has been shown to be a potential immunodiagnostic marker as well as a novel target for chemotherapy. We have cloned, overexpressed and immunochemically characterized the recombinant lactate dehydrogenase of Plasmodium knowlesi, the fifth human malaria parasite. The P. knowlesi lactate dehydrogenase (PkLDH) gene was PCR amplified and 0.9 kb PCR product was cloned into pGEM-T Easy vector. Sequencing and BLAST analysis revealed open reading frame of 316 amino acids of PkLDH showing 96.8% homology with Plasmodium vivax LDH and around 90% with Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale LDHs. The PkLDH gene was subcloned into pGEX-6P1 expression vector and the SDS-PAGE analysis revealed that about 70% of fusion protein was present in the soluble fraction. The fusion protein was cleaved with PreScission protease and recombinant PkLDH (34 kDa) was affinity purified to homogeneity. The purified PkLDH exhibited high reactivity with polyclonal and monoclonal antibodies against plasmodial LDH. The polyclonal antibody produced against purified recombinant PkLDH in rabbits showed high ELISA reactivity with both native and recombinant PkLDH and could detect parasite LDH in malaria infected blood samples by sandwich ELISA. The purified recombinant PkLDH can be used to produce P. knowlesi specific monoclonal antibodies for specific diagnosis of P. knowlesi infection in humans.     

Plasmodium vevax and P. falciparum: Biological interactions and the possibility of cross-species immunity

The question of whether infection of humans with one species of malaria parasite alters the course of infection with another has been largely ignored because no such interaction was found during studies of induced malaria in patients with neurosyphilis. However, in animal model systems some degree of cross-species interaction is the rule rather than the exception. Furthermore, recent epidemiological observations in Vanuatu in the South Pacific have suggested a biological interaction between the dominant species, Plasmodium vivax, and P. falciparum. Kathryn Maitland, Tom Williams and Chris Newbold here speculate on the basis of these observations and other published findings that infection with P. vivax may result in the development of immunity sufficient to ameliorate the clinical course of subsequent infections with the potentially lethal parasite P. falciparum.     

Plasmodium malariae and Plasmodium ovale--the "bashful" malaria parasites

Although Plasmodium malariae was first described as an infectious disease of humans by Golgi in 1886 and Plasmodium ovale identified by Stevens in 1922, there are still large gaps in our knowledge of the importance of these infections as causes of malaria in different parts of the world. They have traditionally been thought of as mild illnesses that are caused by rare and, in case of P. ovale, short-lived parasites. However, recent advances in sensitive PCR diagnosis are causing a re-evaluation of this assumption. Low-level infection seems to be common across malaria-endemic areas, often as complex mixed infections. The potential interactions of P. malariae and P. ovale with Plasmodium falciparum and Plasmodium vivax might explain some basic questions of malaria epidemiology, and understanding these interactions could have an important influence on the deployment of interventions such as malaria vaccines.     

Cross-reactive anti-PfCLAG9 antibodies in the sera of asymptomatic parasite carriers of Plasmodium vivax

The PfCLAG9 has been extensively studied because their immunogenicity. Thereby, the gene product is important for therapeutics interventions and a potential vaccine candidate. Antibodies against synthetic peptides corresponding to selected sequences of the Plasmodium falciparum antigen PfCLAG9 were found in sera of falciparum malaria patients from Rondônia, in the Brazilian Amazon. Much higher antibody titres were found in semi-immune and immune asymptomatic parasite carriers than in subjects suffering clinical infections, corroborating original findings in Papua Guinea. However, sera of Plasmodium vivax patients from the same Amazon area, in particular from asymptomatic vivax parasite carriers, reacted strongly with the same peptides. Bioinformatic analyses revealed regions of similarity between P. falciparum Pfclag9 and the P. vivax ortholog Pvclag7. Indirect fluorescent microscopy analysis showed that antibodies against PfCLAG9 peptides elicited in BALB/c mice react with human red blood cells (RBCs) infected with both P. falciparum and P. vivax parasites. The patterns of reactivity on the surface of the parasitised RBCs are very similar. The present observations support previous findings that PfCLAG9 may be a target of protective immune responses and raises the possibility that the cross reactive antibodies to PvCLAG7 in mixed infections play a role in regulate the fate of Plasmodium mixed infections.     

Performance of OptiMAL(R) in the diagnosis of Plasmodium vivax and Plasmodium falciparum infections in a malaria referral center in Colombia

Alternative, non-microscopic methods for the diagnosis of malaria have recently become available. Among these, rapid dipstick methods stand out. One such test, OptiMAL(R), is based on the immunochromatographic detection of Plasmodium lactate dehydrogenase (pLDH) and has the capacity to detect and distinguish infections caused by P. falciparum and Plasmodium sp. This capacity is particularly important in countries where different species of Plasmodium co-exist. In this study we evaluated the performance of OptiMAL(R) in an urban referral center for malaria diagnosis. Two sets of patients were included: one (n = 112) having predetermined infections with P. falciparum or P. vivax and individuals with negative blood smears; and another consisting of all eligible consecutive patients (n = 80) consulting for diagnosis at the referral center during one month. The overall diagnostic efficiency of OptiMAL(R) for both sets of patients was 96.9%. Efficiency was higher for P. vivax (98.1%) than for P. falciparum (94.9%). These results corroborate the diagnostic utility of OptiMAL(R) in settings where P. vivax and P. falciparum co-exist and support its implementation where microscopic diagnosis is unavailable and in circumstances that exceed the capacity of the local microscopic diagnosis facility.     

The Plasmodium vivax genome sequencing project

With the successful completion of the project to sequence the Plasmodium falciparum genome, researchers are now turning their attention to other malaria parasite species. Here, an update on the Plasmodium vivax genome sequencing project is presented, as part of the Trends in Parasitology series of reviews expanding on various aspects of P. vivax research.     

High rate of detection of mixed infections of Plasmodium vivax and Plasmodium falciparum in South-East of Iran, using nested PCR

Sistan and Baluchestan province, South-East of Iran, has been reported as an endemic area of malaria [Sadrizadeh B. Malaria in the world, in the eastern Mediterranean region and in Iran: Review article. WHO/EMRO Report 2001: 1-13.]. The main objective of this research was to perform rapid and correct diagnoses of malaria infection. Blood specimens were collected from 140 suspected volunteers. The Giemsa-stained slides examination and nested PCR for amplification of the Plasmodium small subunit ribosomal genes (ssrRNA) were utilized. The results demonstrated 118 out of 140 cases (84.3%) positive for malaria parasites, including 60.7%, 20.7% and 2.9% as having Plasmodium vivax (P.v), Plasmodium falciparum (P.f) and mixed infections (P.v+P.f), respectively by microscopy. The nested PCR detected malaria parasites in 134 samples (94.3%), consisting of 51.4% P.v, 12.6% P.f and 29.3% mixed infections. The PCR analysis detected 37 cases of mixed infections more than that of the routine microscopy. These results suggested that there are a considerable number of cases with mixed infections in the study area that mainly remain undiagnosed by microscopy. It is also concluded that the nested PCR is a suitable complement to microscopy for accurate specific diagnosis of malaria species in field.     

Usefulness of genus-specific PCR and Southern blot species-specific hybridization for the detection of imported malaria cases in Italy

A PCR method involving a genus-specific oligonucleotides set and Southern blot hybridization with four species-specific probes to P. falciparum, P. vivax, P. malariae and P. ovale was evaluated for the detection of malaria parasites in blood samples from 101 patients with clinically suspect malaria infection imported to Italy. Plasmodium falciparum was the main species detected. As determined by microscopy, 53 (52.4%) patients had malaria and of these: 40 (75.5%) were infected with P. falciparum; 7 (13.2%) with P. vivax; 1 (1.9%) with P. ovale; 3 (5.7%) with P. malariae; 1 (1.9%) with P. vivax or P. ovale; and 1 (1.9%) with P. falciparum or P. vivax. Ninety-seven out 101 blood samples were submitted to ParaSight-F test which showed a sensitivity of 94.73%, and a specificity of 93.22%, as compared to microscopy. The PCR assay using the genus-specific oligonucleotide primer set (pg-PCR) was able to detect 53 (52.4%) infections and showed a sensitivity of 100% and a specificity of 100%, when compared to microscopy. The parasite species were identified by Southern blot hybridization using species-specific probes and 40 (75.5%) samples were P. falciparum positive, 5 (9.4%) P. vivax positive, 4 (7.5%) P. ovale positive, and 2 (3.8%) P. malariae positive. When the Southern blot results were compared to those of blood-film diagnosis, we observed some disagreement. In particular, compared to Southern blot, microscopy underestimated P. ovale infection; blood film analysis recognised only 1 P. ovale sample, whereas Southern blot recognised 4 P. ovale positive samples (by microscopy, 2 of these were detected as P. vivax, 1 as P. ovale or P. vivax, and the other as P. falciparum or P. vivax). Southern blot hybridization was unable to identify one P. falciparum and one P. vivax positive case detected by microscopy. We also plan to use a reference nested-PCR assay to clarify the disagreement observed between microscopy and Southern blot hybridization.     

Pv12, a 6-Cys antigen of Plasmodium vivax, is localized to the merozoite rhoptry

Pf12 in Plasmodium falciparum has been characterized as a merozoite surface protein and the Pf12 gene is actively transcribed during schizont stage. An orthologous gene, Pv12, has been identified in genome of P. vivax, but the protein product has not been characterized. The Pv12 is a 362 amino acid long polypeptide encoded by a single exon gene PVX_113775, for which orthologous genes have been identified in other Plasmodium species by bioinformatic approaches. Pv12 contains two predicted six-cysteine (6-Cys) domains, which may be constrained by predicted disulfide bonds, and a transmembrane domain and a predicted GPI anchor attachment site in C-terminal region. The recombinant Pv12 protein is recognized by serum antibodies of patients naturally exposed to P. vivax and the native Pv12 protein from parasite extract is also recognized by immune mouse serum. The Pv12 is localized in rhoptry; an apical organelle of the merozoite, and the localization pattern of Pv12 is distinct from that of Pf12 in P. falciparum. The present study suggests that Pv12 is immunogenic in humans during parasite infection and it could play an important role in erythrocyte invasion.     

The Plasmodium vivax rhoptry-associated protein 1

Rhoptries are cellular organelles localized at the apical pole of apicomplexan parasites. Their content is rich in lipids and proteins that are released during target cell invasion. Plasmodium falciparum rhoptry-associated protein 1 (RAP1) has been the most widely studied among this parasite species' rhoptry proteins and is considered to be a good anti-malarial vaccine candidate since it displays little polymorphism and induces antibodies in infected humans. Monoclonal antibodies directed against RAP1 are also able to inhibit target cell invasion in vitro and protection against P. falciparum experimental challenge is induced when non-human primates are immunized with this protein expressed in its recombinant form. This study describes identifying and characterizing RAP1 in Plasmodium vivax, the most widespread parasite species causing malaria in humans, producing more than 80 million infections yearly, mainly in Asia and Latin America. This new protein is encoded by a two-exon gene, is proteolytically processed in a similar manner to its falciparum homologue and, as observed by microscopy, the immunofluorescence pattern displayed is suggestive of its rhoptry localization. Further studies evaluating P. vivax RAP1 protective efficacy in non-human primates should be carried out taking into account the relevance that its P. falciparum homologue has as an anti-malarial vaccine candidate.     

Codon usage in Plasmodium vivax nuclear genes.

Codon usage in Plasmodium vivax nuclear genes was analysed and compared with that in Plasmodium falciparum nuclear genes. Preferred codons were determined for P. vivax. Unlike P. falciparum, P. vivax genes are about 15% less A+T rich in the coding regions, with no obvious A+T bias at the third position of the codons. The amino-acid composition of P. vivax gene products is also different from that of P. falciparum. These results provide valuable information to facilitate gene cloning as well as expression and transfection studies for P. vivax.     

Specific detection of Plasmodium falciparum malaria by a molecularly cloned DNA probe

A highly repeated DNA sequence from Plasmodium falciparum was cloned and used as a probe in molecular hybridization to detect malaria. Our results indicate that the probe is specific to P. falciparum but not to other species of Plasmodium and is extremely sensitive. As little as a 20 pg parasite DNA, which is equivalent to about 1000 parasites can be detected. The cloned DNA can be used as a diagnostic tool to follow the course of infection of falciparum malaria.