[show abstract][hide abstract] ABSTRACT: 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.
The Journal of Infectious Diseases 01/2011; 203(2):207-10. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Plasmodium species ex vivo sensitivity assay protocols differ in the requirement for leukocyte removal before culturing. This study shows that the presence of leukocytes significantly increases the 50% inhibitory concentration (IC₅₀) of P. vivax and P. falciparum to artesunate and chloroquine relative to results with the paired leukocyte-free treatment. Although leukocyte removal is not an essential requirement for the conduct of ex vivo assays, its use has important implications for the interpretation of temporal and spatial antimalarial sensitivity data.
Antimicrobial Agents and Chemotherapy 12/2010; 55(3):1300-4. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: In order to characterize A/H5N1 viral sequences, a bioinformatics approach accurately identified viral sequences from discovery of a sequence signature, which provided enough distinctive information for sequence identification. Eight highly pathogenic H5N1 viral isolations were collected from different areas of Thailand between 2003 and 2006, and were used for analysis of H5N1 genotypic testing with a semiconductor-based oligonucleotide microarray. All H5N1 samples and H1N1, H4N8 negative controls were correctly subtyped. Sensitivity of the eight oligonucleotide probes, with optimized cut-offs, ranged from 70% (95% CI 65-75) to 87% (95% CI 84-91), and the corresponding Kappa values ranged from 0.76 (95% CI 0.72-0.80) to 0.86 (95% CI 0.83-0.89). Semi-conductor-based oligonucleotide array and oligonucleotide probes corresponded well when detecting H5N1. After fully correcting the subtype from the result of microarray signal intensity, the microarray output method combined with bioinformatics tools, identified and monitored genetic variations of H5N1. Capability of distinguishing different strains of H5N1 from Thailand was the outstanding feature of this assay. Ninety percent of HA and NA (4/5) genes were sequenced correctly, in accordance with previous examinations performed by classical diagnostic methods. The low-medium-high bioinformatics resolutions were able to predict an epidemic strain of H5N1. This study also showed the advantage of using a large genotypic database to predict the epidemic strain of H5N1. However, the monitoring protocol of this new strain has been recommended for further study with a large-scale sample.
Molecular and Cellular Probes 12/2010; 24(6):387-95. · 1.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: The novel organometallic chloroquine analog ferroquine (SSR 97193) is effective against chloroquine-resistant Plasmodium falciparum. The ex vivo efficacy of ferroquine against Plasmodium vivax isolates was tested. Ferroquine has a potent ex vivo effect on P. vivax schizont maturation (median 50% inhibitory concentration, 15 nM; n = 42). No significant cross-sensitivity between ferroquine and other antimalarials was detected. This drug may be a suitable replacement for chloroquine in the treatment of drug-resistant P. vivax malaria.
Antimicrobial Agents and Chemotherapy 03/2010; 54(5):2228-30. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: Investigations of Plasmodium vivax are restricted to samples collected from infected persons or primates, because this parasite cannot be maintained in in vitro cultures. Contamination of P. vivax isolates with host leukocytes and platelets is detrimental to a range of ex vivo 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 P. vivax IRBCs by CF11 cellulose filtration.
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 x 103 per microl [95%CI 5.2-13.5] to 0.01 x 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 x 103 per microl [95%CI 107.5-315.7] to 0.8 x 103 per microl [95%CI -0.7-2.2]. Analysis of 30 P. vivax blood samples before and after CF11 filtration showed only a minor loss in parasitaemia (<or= 7.1% of initial counts). Stage specific retention of P. vivax IRBCs was not observed.
CF11 filtration is the most cost and time efficient method for the production of leukocyte- and platelet-free P. vivax-infected erythrocytes from field isolates.
[show abstract][hide abstract] ABSTRACT: The aim of this study was to determine the performance of real-time amplification based methods - NASBA, TaqMan, RT-FRET, and RT-PCR LUXtrade mark formats - for the detection of influenza A (H5N1) virus RNA. In an analysis of 54 samples obtained from a range of animal species in Thailand during the period 2003-2006, results showed that the NASBA (H5=98.2%, N1=96.3%), TaqMan (H5=98.2%, N1=96.3%) and FRET (H5=98.2%, N1=96.3%) had significantly higher rates of positive detection than LUX (H5=94.4%, N1=50.0%; P<0.001) for influenza A, H5 and N1 isolates. There were no false-positive results from any methods used in the negative-control group of samples. The limits of analytical detection were at least 10copies/reaction in real-time NASBA and LUX assays, while FRET and TaqMan assay appeared to be less sensitive at > or =100copies/reaction. The assays were relatively specific without cross-reactivity to a number of other influenza strains or viral pathogens. In conclusion, our study demonstrated that real-time NASBA, TaqMan and FRET assays can be used to detect influenza A (H5N1) from a wide range of hosts, and be specific for H5N1 samples obtained during different outbreaks (2003-2006). All assays provided the benefit of rapid influenza H5N1 identification for early diagnosis, in the range of hours, and they are well suited to high throughput analyses.
Molecular and Cellular Probes 07/2008; 22(5-6):287-93. · 1.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: Plasmodium spp. ex vivo sensitivity assay protocols differ in the requirement for leukocyte removal before culturing. This study shows that the presence of leukocytes significantly increase the IC50 of P. vivax and P. falciparum to artesunate and chloroquine relative to the paired leukocyte free treatment. Although leukocyte removal is not an essential requirement for the conduct of ex vivo assays, its use has important implications for the interpretation of temporal and spatial antimalarial sensitivity data.