Comparison of three molecular methods for the detection and speciation of Plasmodium vivax and Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>Accurate diagnosis of <it>Plasmodium </it>spp. is essential for the rational treatment of malaria. Despite its many disadvantages, microscopic examination of blood smears remains the current "gold standard" for malaria detection and speciation. PCR assays offer an alternative to microscopy which has been shown to have superior sensitivity and specificity. Unfortunately few comparative studies have been done on the various molecular based speciation methods.</p> <p>Methods</p> <p>The sensitivity, specificity and cost effectiveness of three molecular techniques were compared for the detection and speciation of <it>Plasmodium falciparum </it>and <it>Plasmodium vivax </it>from dried blood spots collected from 136 patients in western Thailand. The results from the three molecular speciation techniques (nested PCR, multiplex PCR, and real-time PCR) were used to develop a molecular consensus (two or more identical PCR results) as an alternative gold standard.</p> <p>Results</p> <p>According to the molecular consensus, 9.6% (13/136) of microscopic diagnoses yielded false negative results. Multiplex PCR failed to detect <it>P. vivax </it>in three mixed isolates, and the nested PCR gave a false positive <it>P. falciparum </it>result in one case. Although the real-time PCR melting curve analysis was the most expensive method, it was 100% sensitive and specific and least time consuming of the three molecular techniques investigated.</p> <p>Conclusion</p> <p>Although microscopy remains the most appropriate method for clinical diagnosis in a field setting, its use as a gold standard may result in apparent false positive results by superior techniques. Future studies should consider using more than one established molecular methods as a new gold standard to assess novel malaria diagnostic kits and PCR assays.</p

Methotrexate Is Highly Potent Against Pyrimethamine-Resistant Plasmodium vivax

Resistance of vivax malaria to treatment with antifolates, such as pyrimethamine (Pyr), is spreading as mutations in the dihydrofolatereductase (dhfr) genes are selected and disseminated. We tested the antitumor drug methotrexate (MTX), a potent competitive inhibitor of dhfr, against 11 Plasmodium vivax isolates ex vivo, 10 of which had multiple dhfr mutations associated with Pyr resistance. Despite high-grade resistance to Pyr (median 50% inhibitory concentration [IC50], 13,345 nM), these parasites were all highly susceptible to MTX (median IC50, 2.6 nM). Given its potency against Pyr-resistant P. vivax, the antimalarial potential of MTX deserves further investigation

Effective and cheap removal of leukocytes and platelets from Plasmodium vivax infected blood

<p>Abstract</p> <p>Background</p> <p>Investigations of <it>Plasmodium vivax </it>are restricted to samples collected from infected persons or primates, because this parasite cannot be maintained in <it>in vitro </it>cultures. Contamination of <it>P. vivax </it>isolates with host leukocytes and platelets is detrimental to a range of <it>ex vivo </it>and molecular investigations. Easy-to-produce CF11 cellulose filters have recently provided us with an inexpensive method for the removal of leukocytes and platelets. This contrasted with previous reports of unacceptably high levels of infected red blood cell (IRBC) retention by CF11. The aims of this study were to compare the ability of CF11 cellulose filters and the commercial filter Plasmodipur at removing leukocyte and platelet, and to investigate the retention of <it>P. vivax </it>IRBCs by CF11 cellulose filtration.</p> <p>Methods and Results</p> <p>Side-by-side comparison of six leukocyte removal methods using blood samples from five healthy donor showed that CF11 filtration reduced the mean initial leukocyte counts from 9.4 × 10³per μl [95%CI 5.2–13.5] to 0.01 × 10³[95%CI 0.01–0.03]. The CF11 was particularly effective at removing neutrophils. CF11 treatment also reduced initial platelet counts from 211.6 × 10³per μl [95%CI 107.5–315.7] to 0.8 × 10³per μl [95%CI -0.7–2.2]. Analysis of 30 <it>P. vivax </it>blood samples before and after CF11 filtration showed only a minor loss in parasitaemia (≤ 7.1% of initial counts). Stage specific retention of <it>P. vivax </it>IRBCs was not observed.</p> <p>Conclusion</p> <p>CF11 filtration is the most cost and time efficient method for the production of leukocyte- and platelet-free <it>P. vivax</it>-infected erythrocytes from field isolates.</p

Considerations on the use of nucleic acid-based amplification for malaria parasite detection

<p>Abstract</p> <p>Background</p> <p>Nucleic acid amplification provides the most sensitive and accurate method to detect and identify pathogens. This is primarily useful for epidemiological investigations of malaria because the infections, often with two or more <it>Plasmodium </it>species present simultaneously, are frequently associated with microscopically sub-patent parasite levels and cryptic mixed infections. Numerous distinct equally adequate amplification-based protocols have been described, but it is unclear which to select for epidemiological surveys. Few comparative studies are available, and none that addresses the issue of inter-laboratory variability.</p> <p>Methods</p> <p>Blood samples were collected from patients attending malaria clinics on the Thai-Myanmar border. Frozen aliquots from 413 samples were tested independently in two laboratories by nested PCR assay. Dried blood spots on filter papers from the same patients were also tested by the nested PCR assay in one laboratory and by a multiplex PCR assay in another. The aim was to determine which protocol best detected parasites below the sensitivity level of microscopic examination.</p> <p>Results</p> <p>As expected PCR-based assays detected a substantial number of infected samples, or mixed infections, missed by microscopy (27 and 42 for the most sensitive assay, respectively). The protocol that was most effective at detecting these, in particular mixed infections, was a nested PCR assay with individual secondary reactions for each of the species initiated with a template directly purified from the blood sample. However, a lesser sensitivity in detection was observed when the same protocol was conducted in another laboratory, and this significantly altered the data obtained on the parasite species distribution.</p> <p>Conclusions</p> <p>The sensitivity of a given PCR assay varies between laboratories. Although, the variations are relatively minor, they primarily diminish the ability to detect low-level and mixed infections and are sufficient to obviate the main rationale to use PCR assays rather than microscopy or rapid diagnostic tests. The optimal approach to standardise methodologies is to provide PCR template standards. These will help researchers in different settings to ensure that the nucleic acid amplification protocols they wish to use provide the requisite level of sensitivity, and will permit comparison between sites.</p

Plasmodium vivax trophozoites insensitive to chloroquine

© 2008 Sharrock et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Plasmodium vivax lineages: geographical distribution, tandem repeat polymorphism, and phylogenetic relationship

Background: Multi-drug resistance and severe/ complicated cases are the emerging phenotypes of vivax malaria, which may deteriorate current anti-malarial control measures. The emergence of these phenotypes could be associated with either of the two Plasmodium vivax lineages. The two lineages had been categorized as Old World and New World, based on geographical sub-division and genetic and phenotypical markers. This study revisited the lineage hypothesis of P. vivax by typing the distribution of lineages among global isolates and evaluated their genetic relatedness using a panel of new mini-satellite markers. Methods: 18S SSU rRNA S-type gene was amplified from 420 Plasmodium vivax field isolates collected from different geographical regions of India, Thailand and Colombia as well as four strains each of P. vivax originating from Nicaragua, Panama, Thailand (Pak Chang), and Vietnam (ONG). A mini-satellite marker panel was then developed to understand the population genetic parameters and tested on a sample subset of both lineages. Results: 18S SSU rRNA S-type gene typing revealed the distribution of both lineages (Old World and New World) in all geographical regions. However, distribution of Plasmodium vivax lineages was highly variable in every geographical region. The lack of geographical sub-division between lineages suggests that both lineages are globally distributed. Ten mini-satellites were scanned from the P. vivax genome sequence; these tandem repeats were located in eight of the chromosomes. Mini-satellites revealed substantial allelic diversity (7-21, AE = 14.6 +/- 2.0) and heterozygosity (He = 0.697-0.924, AE = 0.857 +/- 0.033) per locus. Mini-satellite comparison between the two lineages revealed high but similar pattern of genetic diversity, allele frequency, and high degree of allele sharing. A Neighbour-Joining phylogenetic tree derived from genetic distance data obtained from ten mini-satellites also placed both lineages together in every cluster. Conclusions: The global lineage distribution, lack of genetic distance, similar pattern of genetic diversity, and allele sharing strongly suggested that both lineages are a single species and thus new emerging phenotypes associated with vivax malaria could not be clearly classified as belonging to a particular lineage on basis of their geographical origin

Genetic Diversity in New Members of the Reticulocyte Binding Protein Family in Thai Plasmodium vivax Isolates

Background Plasmodium vivax merozoites specifically invade reticulocytes. Until recently, two reticulocyte-binding proteins (Pvrbp1 and Pvrbp2) expressed at the apical pole of the P. vivax merozoite were considered to be involved in reticulocyte recognition. The genome sequence recently obtained for the Salvador I (Sal-I) strain of P. vivax revealed additional genes in this family, and in particular Pvrbp2a, Pvrbp2b (Pvrbp2 has been renamed as Pvrbp2c) and two pseudogenes Pvrbp2d and Pvrbp3. It had been previously found that Pvrbp2c is substantially more polymorphic than Pvrbp1. The primary goal of this study was to ascertain the level of polymorphism of these new genes. Methodology/Principal Findings The sequence of the Pvrbp2a, Pvrbp2b, Pvrbp2d and Pvrbp3 genes were obtained by amplification/cloning using DNA purified from four isolates collected from patients that acquired the infection in the four cardinal regions of Thailand (west, north, south and east). An additional seven isolates from western Thailand were analyzed for gene copy number variation. There were significant polymorphisms exhibited by these genes (compared to the reference Sal-I strain) with the ratio of mutations leading to a non-synonymous or synonymous amino acid change close to 3∶1 for Pvrbp2a and Pvrbp2b. Although the degree of polymorphism exhibited by these two genes was higher than that of Pvrbp1, it did not reach the exceptional diversity noted for Pvrbp2c. It was interesting to note that variations in the copy number of Pvrbp2a and Pvrbp2b occurred in some isolates. Conclusions/Significance The evolution of different members of the Pvrbp2 family and their relatively high degree of polymorphism suggests that the proteins encoded by these genes are important for parasite survival and are under immune selection. Our data also shows that there are highly conserved regions in rbp2a and rbp2b, which might provide suitable targets for future vaccine development against the blood stage of P. vivax

In Vivo and In Vitro Activities and ADME-Tox Profile of a Quinolizidine-Modified 4-Aminoquinoline: A Potent Anti-P. falciparum and Anti-P. vivax Blood-Stage Antimalarial.

Natural products are a prolific source for the identification of new biologically active compounds. In the present work, we studied the in vitro and in vivo antimalarial efficacy and ADME-Tox profile of a molecular hybrid (AM1) between 4-aminoquinoline and a quinolizidine moiety derived from lupinine (Lupinus luteus). The aim was to find a compound endowed with the target product profile-1 (TCP-1: molecules that clear asexual blood-stage parasitaemia), proposed by the Medicine for Malaria Venture to accomplish the goal of malaria elimination/eradication. AM1 displayed a very attractive profile in terms of both in vitro and in vivo activity. By using standard in vitro antimalarial assays, AM1 showed low nanomolar inhibitory activity against chloroquine-sensitive and resistant P. falciparum strains (range IC50 16-53 nM), matched with a high potency against P. vivax field isolates (Mean IC50 29 nM). Low toxicity and additivity with artemisinin derivatives were also demonstrated in vitro. High in vivo oral efficacy was observed in both P.berghei and P. yoelii mouse models with IC50 values comparable or better than those of chloroquine. The metabolic stability in different species and the pharmacokinetic profile in the mouse model makes AM1 a compound worth further investigation as a potential novel schizonticidal agent

Significant Biochemical, Biophysical and Metabolic Diversity in Circulating Human Cord Blood Reticulocytes

10.1371/journal.pone.0076062PLoS ONE810-POLN