Improvement of culture conditions for long-term in vitro culture of Plasmodium vivax

Roobsoong, Wanlapa; Tharinjaroen, Chayada Sitthidet; Rachaphaew, Nattawan; Chobson, Porpimon; Schofield, Louis; Cui, Liwang; Adams, John; Sattabongkot, Jetsumon

doi:10.1186/s12936-015-0815-z

Cited by 44 publications

(65 citation statements)

References 21 publications

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“…Many culture medium formulations have been reported for the ex vivo maturation of P. vivax (4,7,18,19). Most current formulations are based on McCoy's 5A medium with human serum added with or without additional supplements.…”

Section: Discussionmentioning

confidence: 99%

Enhanced Ex Vivo Plasmodium vivax Intraerythrocytic Enrichment and Maturation for Rapid and Sensitive Parasite Growth Assays

Rangel

Clark

Kanjee

et al. 2018

Antimicrob Agents Chemother

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chloroquine resistance has been documented in nearly every region where this malaria-causing parasite is endemic. Unfortunately, resistance surveillance and drug discovery are challenging due to the low parasitemias of patient isolates and poor parasite survival through maturation that reduce the sensitivity and scalability of current antimalarial assays. Using cryopreserved patient isolates from Brazil and fresh patient isolates from India, we established a robust enrichment method for parasites. We next performed a medium screen for formulations that enhance survival. Finally, we optimized an isotopic metabolic labeling assay for measuring maturation and its sensitivity to antimalarials. A KCl Percoll density gradient enrichment method increased parasitemias from small-volume isolates by an average of>40-fold. The use of Iscove's modified Dulbecco's medium for culture approximately doubled the parasite survival through maturation. Coupling these with [H]hypoxanthine metabolic labeling permitted sensitive and robust measurements of parasite maturation, which was used to measure the sensitivities of Brazilian isolates to chloroquine and several novel antimalarials. These techniques can be applied to rapidly and robustly assess the isolate sensitivities to antimalarials for resistance surveillance and drug discovery.

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Section: Discussionmentioning

confidence: 99%

Enhanced Ex Vivo Plasmodium vivax Intraerythrocytic Enrichment and Maturation for Rapid and Sensitive Parasite Growth Assays

Rangel

Clark

Kanjee

et al. 2018

Antimicrob Agents Chemother

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“…One study demonstrated robust invasion assay which has allowed testing molecules in invasion of 2C3 as the monoclonal antibody of DARC for blocked PvDBP interaction by short term P. vivax invasion process 13 . An invasion mechanism study of P. vivax faces a considerable hurdle owing to the inability to continuously culture the parasites in vitro 14 15 . To overcome these problems, we consider evidence from homologous protein families in other Plasmodium spp.…”

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confidence: 99%

Identification of a reticulocyte-specific binding domain of Plasmodium vivax reticulocyte-binding protein 1 that is homologous to the PfRh4 erythrocyte-binding domain

Han

Lee

Wang

et al. 2016

Sci Rep

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The Plasmodium vivax reticulocyte-binding protein (RBP) family was identified based on the annotation of adhesive ligands in the P. vivax genome. Reticulocyte-specific interactions with the PvRBPs (PvRBP1 and PvRBP2) were previously reported. Plasmodium falciparum reticulocyte-binding protein homologue 4 (PfRh4, a homologue of PvRBP1) was observed to possess erythrocyte-binding activity via complement receptor 1 on the erythrocyte surface. However, the reticulocyte-binding mechanisms of P. vivax are unclear because of the large molecular mass of PvRBP1 (>326 kDa) and the difficulty associated with in vitro cultivation. In the present study, 34 kDa of PvRBP1a (PlasmoDB ID: PVX_098585) and 32 kDa of PvRBP1b (PVX_098582) were selected from a 30 kDa fragment of PfRh4 for reticulocyte-specific binding activity analysis. Both PvRBP1a and PvRBP1b were found to be localized at the microneme in the mature schizont-stage parasites. Naturally acquired immune responses against PvRBP1a-34 and PvRBP1b-32 were observed lower than PvDBP-RII. The reticulocyte-specific binding activities of PvRBP1a-34 and PvRBP1b-32 were significantly higher than normocyte binding activity and were significantly reduced by chymotrypsin treatment. PvRBP1a and 1b, bind to reticulocytes and that this suggests that these ligands may have an important role in P. vivax merozoite invasion.

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“…While there have been some recent improvements to in vitro culture techniques for Plasmodium vivax, the culture of this parasite is still limited by the requirement of reticulocytes, and the parasite density over longterm culture is low (7). However, the recent adaptation of the zoonotic malaria species Plasmodium knowlesi (8) to continuous in vitro culture in human erythrocytes (9)(10)(11) has changed this position.…”

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confidence: 99%

Adaptation of the [ ³ H]Hypoxanthine Uptake Assay for In Vitro -Cultured Plasmodium knowlesi Malaria Parasites

Arnold

Engel

Chua

et al. 2016

Antimicrob Agents Chemother

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The zoonotic malaria parasite Plasmodium knowlesi has recently been established in continuous in vitro culture. Here, the Plasmodium falciparum [ 3 H]hypoxanthine uptake assay was adapted for P. knowlesi and used to determine the sensitivity of this parasite to chloroquine, cycloguanil, and clindamycin. The data demonstrate that P. knowlesi is sensitive to all drugs, with 50% inhibitory concentrations (IC 50 s) consistent with those obtained with P. falciparum. This assay provides a platform to use P. knowlesi in vitro for drug discovery. In 2015, there were an estimated 214 million clinical cases of malaria which resulted in ϳ438,000 deaths (1). Substantial funds have been invested in producing a malaria vaccine; however, the efficacy of experimental vaccines has been poor (2, 3), and as a result, vector control and drugs remain the mainstays for the prevention and treatment of malaria. While there has been a significant reduction in malaria-associated mortality and morbidity in recent years (1), there is concern that lack of sustained funding, together with insecticide and antimalarial drug resistance, will affect this progress (4). To prevent backward momentum in disease control and push toward the endgame strategy of malaria elimination, new chemotherapeutics with novel modes of action and activity against multiple species and life cycle stages are needed (5).The ability to easily and rapidly assess the activity of new lead compounds against multiple Plasmodium species has been limited to date, as only Plasmodium falciparum has been amenable to routine, long-term, continuous in vitro culture (6). While there have been some recent improvements to in vitro culture techniques for Plasmodium vivax, the culture of this parasite is still limited by the requirement of reticulocytes, and the parasite density over longterm culture is low (7). However, the recent adaptation of the zoonotic malaria species Plasmodium knowlesi (8) to continuous in vitro culture in human erythrocytes (9-11) has changed this position. Although a routine drug sensitivity assay for P. knowlesi has not yet been established, the ability to culture this parasite species in vitro provides researchers with an unprecedented opportunity to rapidly test new drug leads against two human-infecting Plasmodium species.In vitro assays for assessing malaria parasite growth inhibition are indispensable tools for the screening and evaluation of potential new drug leads and, also, for the surveillance of parasite drug resistance. A "gold standard" approach for assessing P. falciparum growth inhibition is the incorporation of [ 3 H]hypoxanthine into parasite nucleic acids (12). As Plasmodium parasites are unable to synthesize purines de novo, they must scavenge these metabolic precursors for growth. Thus, supplementation of parasite cultures with [3 H]hypoxanthine results in the incorporation of this radiolabeled purine into nucleic acids, permitting growth to be quantitated using a scintillation counter. While there are a number of other methods available t...

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Improvement of culture conditions for long-term in vitro culture of Plasmodium vivax

Cited by 44 publications

References 21 publications

Enhanced Ex Vivo Plasmodium vivax Intraerythrocytic Enrichment and Maturation for Rapid and Sensitive Parasite Growth Assays

Enhanced Ex Vivo Plasmodium vivax Intraerythrocytic Enrichment and Maturation for Rapid and Sensitive Parasite Growth Assays

Identification of a reticulocyte-specific binding domain of Plasmodium vivax reticulocyte-binding protein 1 that is homologous to the PfRh4 erythrocyte-binding domain

Adaptation of the [ ³ H]Hypoxanthine Uptake Assay for In Vitro -Cultured Plasmodium knowlesi Malaria Parasites

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Improvement of culture conditions for long-term in vitro culture of Plasmodium vivax

Cited by 44 publications

References 21 publications

Enhanced Ex Vivo Plasmodium vivax Intraerythrocytic Enrichment and Maturation for Rapid and Sensitive Parasite Growth Assays

Enhanced Ex Vivo Plasmodium vivax Intraerythrocytic Enrichment and Maturation for Rapid and Sensitive Parasite Growth Assays

Identification of a reticulocyte-specific binding domain of Plasmodium vivax reticulocyte-binding protein 1 that is homologous to the PfRh4 erythrocyte-binding domain

Adaptation of the [ 3 H]Hypoxanthine Uptake Assay for In Vitro -Cultured Plasmodium knowlesi Malaria Parasites

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Adaptation of the [ ³ H]Hypoxanthine Uptake Assay for In Vitro -Cultured Plasmodium knowlesi Malaria Parasites