Optimized In Vitro Production of Plasmodium vivax Ookinetes

Colleen M. McClean Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla, California; Asociación Benéfica PRISMA, Lima, Peru; Institute of Tropical Medicine, Universidad Peruana Cayetano Heredia, Lima, Peru

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Haydee Guerra Alvarado Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla, California; Asociación Benéfica PRISMA, Lima, Peru; Institute of Tropical Medicine, Universidad Peruana Cayetano Heredia, Lima, Peru

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Victor Neyra Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla, California; Asociación Benéfica PRISMA, Lima, Peru; Institute of Tropical Medicine, Universidad Peruana Cayetano Heredia, Lima, Peru

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Alejandro Llanos-Cuentas Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla, California; Asociación Benéfica PRISMA, Lima, Peru; Institute of Tropical Medicine, Universidad Peruana Cayetano Heredia, Lima, Peru

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Joseph M. Vinetz Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla, California; Asociación Benéfica PRISMA, Lima, Peru; Institute of Tropical Medicine, Universidad Peruana Cayetano Heredia, Lima, Peru

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Previous reports have described obtaining mature Plasmodium vivax ookinetes in vitro using blood from infected patients using a simplified, field-based protocol. Here, we report protocols that produce improved P. vivax ookinete yields and morphological development. Optimal conditions included induction of gametogenesis using 10 mM Tris, 170 mM NaCl, 10 mM glucose, 25 mM NaHCO3, and 100 μM xanthurenic acid for 90 minutes at pH 8.0–8.2, followed by culture in RPMI-1640, 50 mg/mL hypoxanthine, 25 mM HEPES, 29 mM NaHCO3, 2 mM L-glutamine, and 20% fetal bovine serum at pH 8.4 for 36 hours. Ookinetes were produced in 86% (18/21) of optimized in vitro cultures; yields ranged from 6.5 × 104 to 2.8 × 106; percent gametocyte conversion ranged from 1.4% to 4.7%. This improved method is suitable for preparation of P. vivax ookinetes in quantities sufficient for biochemical, molecular, and cell biological analysis where basic laboratory facilities are in proximity to patients with vivax malaria.

Author Notes

*Address correspondence to Joseph M. Vinetz, Division of Infectious Diseases, Department of Medicine, University of California San Diego, 9500 Gilman Drive MC 0741, George Palade Laboratories Room 125, La Jolla, CA 92093-0741. E-mail: jvinetz@ucsd.edu

Financial support: This work was supported by U.S. Public Health Service grants 5RO1AI45999, 1D43TW007120, K24AI068903, and R01AI067727.

Authors' addresses: Colleen M. McClean, Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla, CA; current address: PCV, Peace Corps/TOGO, Lomé, Togo, E-mail: cmmcclean@gmail.com. Haydee Guerra Alvarado, Asociación Benéfica PRISMA, Lima, Peru, E-mail: haydeeprisma@gmail.com. Victor Neyra and Alejandro Llanos-Cuentas, Universidad Peruana Cayetano Heredia, Instituto de Medicina Tropical Alexander von Humboldt, Lima, Perú, E-mails: victor.neyra@upch.pe and elmer.llanos@upch.pe. Joseph M. Vinetz, Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla, CA, E-mail: jvinetz@ucsd.edu.

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