World Antimalarial Resistance Network (WARN) II: In vitro antimalarial drug susceptibility

Intrinsic resistance of Plasmodium falciparum is clearly a major determinant of the clinical failure of antimalarial drugs. However, complex interactions between the host, the parasite and the drug obscure the ability to define parasite drug resistance in vivo. The in vitro antimalarial drug susceptibility assay determines ex-vivo growth of parasite in the presence of serial drug concentrations and, thus, eliminates host effects, such as drug metabolism and immunity. Although the sensitivity of the parasite to various antimalarials provided by such a test provides an important indicator of intrinsic parasite susceptibility, there are fundamental methodological issues that undermine comparison of in vitro susceptibility both between laboratories and within a single laboratory over time. A network of laboratories is proposed that will agree on the basic parameters of the in vitro test and associated measures of quality control. The aim of the network would be to establish baseline values of sensitivity to commonly used antimalarial agents from key regions of the world, and create a global database, linked to clinical, molecular and pharmacology databases, to support active surveillance to monitor temporal trends in parasite susceptibility. Such a network would facilitate the rapid detection of strains with novel antimalarial resistance profiles and investigate suitable alternative treatments with retained efficacy

Plasmodium vivax: isotopic, PicoGreen, and microscopic assays for measuring chloroquine sensitivity in fresh and cryopreserved isolates.

In vitro susceptibility tests provide information on the intrinsic response of Plasmodium vivax to antimalarials, free from confounding factors such as host immunity or relapse. This study examined the utility of radioisotope and PicoGreen assays as alternatives to the traditional microscopic examination for assessing response of P. vivax to antimalarial drugs. There was no significant difference in the mean chloroquine IC(50) of P. vivax (n=40) as determined by the microscopic (33.4 ng/ml), isotopic (33.6 ng/ml), and PicoGreen (39.1 ng/ml) assays, respectively (F=0.239, df=2, 51, and p=0.788). However measurement of IC(50)s by the microscopic method was slightly more successful in producing valid assays (57%), compared to the isotopic (32.5%) and PicoGreen (45.5%) methods. In a paired comparison of 20 fresh and cryopreserved isolates as examined by the microscopic method, there were no significant differences between the mean IC(50) responses (T=1.58, df=15, and p=0.34). Detailed methodologies for the short time culture of field and cryopreserved P. vivax are described. Although the microscopic in vitro assay provides a useful method for characterizing the drug susceptibility phenotype of P. vivax isolates, its utility is limited by a laborious methodology and need for highly skilled microscopists. Future efforts should focus on further development of high throughput assays such as the PicoGreen assay as described in this study

Plasmodium vivax: Isotopic, PicoGreen, and microscopic assays for measuring chloroquine sensitivity in fresh and cryopreserved isolates

In vitro susceptibility tests provide information on the intrinsic response of Plasmodium vivax to antimalarials, free from confounding factors such as host immunity or relapse. This study examined the utility of radioisotope and PicoGreen assays as alternatives to the traditional microscopic examination for assessing response of P. vivax to antimalarial drugs. There was no significant difference in the mean chloroquine IC50 of P. vivax (n = 40) as determined by the microscopic (33.4 ng/ml), isotopic (33.6 ng/ml), and PicoGreen (39.1 ng/ml) assays, respectively (F = 0.239, df = 2, 51, and p = 0.788). However measurement of IC50s by the microscopic method was slightly more successful in producing valid assays (57%), compared to the isotopic (32.5%) and PicoGreen (45.5%) methods. In a paired comparison of 20 fresh and cryopreserved isolates as examined by the microscopic method, there were no significant differences between the mean IC50 responses (T = 1.58, df = 15, and p = 0.34). Detailed methodologies for the short time culture of field and cryopreserved P. vivax are described. Although the microscopic in vitro assay provides a useful method for characterizing the drug susceptibility phenotype of P. vivax isolates, its utility is limited by a laborious methodology and need for highly skilled microscopists. Future efforts should focus on further development of high throughput assays such as the PicoGreen assay as described in this study

Unambiguous determination of Plasmodium vivax reticulocyte invasion by flow cytometry

The invasion of CD71+ reticulocytes by Plasmodium vivax is a crucial yet poorly characterised event. The application of flow cytometry to ex vivo invasion assays promises to facilitate the quantitative analysis of P. vivax reticulocyte invasion. However, current protocols suffer from a low level of sensitivity due to the absence of a particular design for P. vivax cell tropism. Importantly, merozoite invasion into contaminating red blood cells from the schizont inoculum (auto-invasion) may confound the analysis. Here we present a stable two-color flow cytometry assay for the accurate quantification of P. vivax merozoite invasion into intracellularly labelled CD71+ reticulocytes. Various enzymatic treatments, antibodies and invasion inhibitory molecules were used to successfully demonstrate the utility of this method. Fluorescent labelling of red blood cells did not affect the invasion and early intra-erythrocytic development of P. vivax. Importantly, this portable field assay allows for the economic usage of limited biological material (parasites and reticulocytes) and the intracellular labeling of the target cells reduces the need for highly purified schizont inoculums. This assay will facilitate the study of P. vivax merozoite biology and the testing of vaccine candidates against vivax malaria

Chloroquine resistant Plasmodium vivax: in vitro characterisation and association with molecular polymorphisms.

BACKGROUND: Treatment failure of chloroquine for P. vivax infections has reached high levels in the eastern provinces of Indonesia, however, in vitro characterization of chloroquine resistance and its associated molecular profile have yet to be determined. METHODS: Using a modified schizont maturation assay we investigated the in vitro chloroquine susceptibility profile and molecular polymorphisms of P. vivax isolates collected from Papua, Indonesia, where high levels of clinical chloroquine treatment failure have been reported, and from Thailand, where chloroquine treatment is generally effective. RESULTS: The geometric mean chloroquine IC(50) for P. vivax isolates from Papua (n = 145) was 312 nM [95%CI: 237-411 nM] compared to 46.8 nM [95%CI: 34.7-63.1 nM] from Thailand (n = 81); p<0.001. Correlating with the known clinical efficacy of the area, a cut off for chloroquine resistance was defined as 220 nM, a level exceeded in 13.6% (11/81) of Thai isolates and 65% (94/145) of Papuan isolates; p<0.001. Several sequence polymorphisms in pvcrt-o and pvmdr1, and difference in pvmdr1 copy number were identified. A Y976F mutation in pvmdr1 was present in 96% (123/128) of Papuan isolates and 25% (17/69) of Thai isolates; p<0.001. Overall, the geometric mean chloroquine IC(50) in isolates with the Y976F mutation was 283 nM [95%CI: 211-379], compared to 44.5 nM [95%CI: 31.3-63.4] in isolates with the wild type; p< 0.001. Pvmdr1 amplification occurred in 23% (15/66) of Thai isolates compared to none (0/104) of Indonesian isolates (p<0.001), but was not associated with increased chloroquine resistance after controlling for geographical location. CONCLUSIONS: In vitro susceptibility testing of P. vivax discriminates between populations with differing levels of clinical efficacy of chloroquine. The pvmdr1 polymorphism at Y976F may provide a useful tool to highlight areas of emerging chloroquine resistance, although further studies defining its clinical correlates are needed

Strict tropism for CD71+/ CD234+ human reticulocytes limits Plasmodium cynomolgi's zoonotic potential

Two malaria parasites of Southeast Asian macaques, Plasmodium knowlesi and P. cynomolgi, can infect humans experimentally. In Malaysia, where both species are common, zoonotic knowlesi malaria has recently become dominant, and cases are recorded throughout the region. By contrast, to date only a single case of naturally acquired P. cynomolgi has been found in humans. In this study we show that whereas P. cynomolgi merozoites invade monkey red blood cells (RBCs) indiscriminately in vitro, for humans they are restricted to reticulocytes expressing both transferrin receptor 1 (Trf1 or CD71) and the Duffy antigen/chemokine receptor (DARC or CD234). This likely contributes to the paucity of detectable zoonotic cynomolgi malaria. We further describe post-invasion morphological and rheological alterations in P. cynomolgi-infected human reticulocytes that are strikingly similar to those observed for P. vivax These observations stress the value of P. cynomolgi as a model in the development of blood stage vaccines against vivax malaria

Strict tropism for CD71+/ CD234+ human reticulocytes limits Plasmodium cynomolgi's zoonotic potential

Two malaria parasites of Southeast Asian macaques, Plasmodium knowlesi and P. cynomolgi, can infect humans experimentally. In Malaysia, where both species are common, zoonotic knowlesi malaria has recently become dominant, and cases are recorded throughout the region. By contrast, to date only a single case of naturally acquired P. cynomolgi has been found in humans. In this study we show that whereas P. cynomolgi merozoites invade monkey red blood cells (RBCs) indiscriminately in vitro, for humans they are restricted to reticulocytes expressing both transferrin receptor 1 (Trf1 or CD71) and the Duffy antigen/chemokine receptor (DARC or CD234). This likely contributes to the paucity of detectable zoonotic cynomolgi malaria. We further describe post-invasion morphological and rheological alterations in P. cynomolgi-infected human reticulocytes that are strikingly similar to those observed for P. vivax These observations stress the value of P. cynomolgi as a model in the development of blood stage vaccines against vivax malaria

Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr) and dihydropteroate synthase (Pvdhps) in P. vivax isolates from malaria endemic areas of Thailand

Malaria is the most important public health problem in several countries. In Thailand, co-infections of Plasmodium vivax and Plasmodium falciparum are common. We examined the prevalence and patterns of mutations in P. vivax dihydrofolate reductase (Pvdhfr) and P. vivax dihydropteroate synthase (Pvdhps) in 103 blood samples collected from patients with P. vivax infection who had attended the malaria clinic in Mae Sot, Tak Province during 2009 and 2010. Using nested polymerase chain reaction-restriction fragment length polymorfism, we examined single nucleotide polymorphisms-haplotypes at amino acid positions 13, 33, 57, 58, 61, 117 and 173 of Pvdhfr and 383 and 553 of Pvdhps. All parasite isolates carried mutant Pvdhfr alleles, of which the most common alleles were triple mutants (99%). Eight different types of Pvdhfr and combination alleles were found, as follows: 57I/58R/117T, 57I/58R/117T, 57I/58R/117T/N, 57L/58R/117T, 57L/58R/117T, 58R/61M/117N, 58R/61M/117N and 13L/57L/58R/117T. The most common Pvdhfr alleles were 57I/58R/117T (77.7%), 57I/58R/117T/N (1%), 57L/58R/117T (5.8%) and 58R/61M/117N (14.5%). The most common Pvdhfr alleles were 57I/58R/117T (77.7%), 57I/58R/117T/N (1%), 57L/58R/117T (5.8%) and 58R/61M/117N (14.5%). Additionally, we recovered one isolate of a carrying a quadruple mutant allele, 13L/57L/58R/117T. The most prevalent Pvdhps allele was a single mutation in amino acid 383 (82.5%), followed by the wild-type A383/A553 (17.5%) allele. Results suggest that all P. vivax isolates in Thailand carry some combination of mutations in Pvdhfr and Pvdhps. Our findings demonstrate that development of new antifolate drugs effective against sulfadoxine-pyrimethamine-resistant P. vivax is required