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

Author:

Rangel Gabriel W.1ORCID,Clark Martha A.1,Kanjee Usheer1,Lim Caeul1,Shaw-Saliba Kathryn1,Menezes Maria José2,Mascarenhas Anjali34,Chery Laura4,Gomes Edwin3,Rathod Pradipsinh K.4,Ferreira Marcelo U.2,Duraisingh Manoj T.1

Affiliation:

1. Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA

2. Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

3. Department of Medicine, Goa Medical College and Hospital, Bambolim, Goa, India

4. Department of Chemistry, University of Washington, Seattle, Washington, USA

Abstract

ABSTRACT Plasmodium vivax chloroquine resistance has been documented in nearly every region where this malaria-causing parasite is endemic. Unfortunately, P. vivax resistance surveillance and drug discovery are challenging due to the low parasitemias of patient isolates and poor parasite survival through ex vivo maturation that reduce the sensitivity and scalability of current P. vivax antimalarial assays. Using cryopreserved patient isolates from Brazil and fresh patient isolates from India, we established a robust enrichment method for P. vivax parasites. We next performed a medium screen for formulations that enhance ex vivo survival. Finally, we optimized an isotopic metabolic labeling assay for measuring P. vivax maturation and its sensitivity to antimalarials. A KCl Percoll density gradient enrichment method increased parasitemias from small-volume ex vivo isolates by an average of >40-fold. The use of Iscove's modified Dulbecco's medium for P. vivax ex vivo culture approximately doubled the parasite survival through maturation. Coupling these with [ 3 H]hypoxanthine metabolic labeling permitted sensitive and robust measurements of parasite maturation, which was used to measure the sensitivities of Brazilian P. vivax isolates to chloroquine and several novel antimalarials. These techniques can be applied to rapidly and robustly assess the P. vivax isolate sensitivities to antimalarials for resistance surveillance and drug discovery.

Funder

Howard Hughes Medical Institute

MCTI | Conselho Nacional de Desenvolvimento Científico e Tecnológico

Gouvernement du Canada | Canadian Institutes of Health Research

Bill and Melinda Gates Foundation

HHS | NIH | National Institute of Allergy and Infectious Diseases

Publisher

American Society for Microbiology

Subject

Infectious Diseases,Pharmacology (medical),Pharmacology

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