Plasmodium vivax liver stage assay platforms using Indian clinical isolates

Pradeep Annamalai Subramani ,Pallavi Mathur,Sowmya Kanchanahalli Basavanna ,Ramya V Krishnamurthy ,Susanta Kumar Ghosh ,Sushrut Dakhore ,Shridhar Narayanan,Seetha Sreekumar,Anupkumar R Anvikar,Kouichi Hasegawa,Radhakrishan Shandil,Benudhar Mukhi,Priyasha Mishra,Neha Vartak-Sharma,Neena Valecha,Noriko Yoshida,Brice Campo,Devaiah Monnanda Kalappa ,Bhavana Nayer,Varadharajan Sundaramurthy

doi:10.1186/s12936-020-03284-8

Abstract

BackgroundVivax malaria is associated with significant morbidity and economic loss, and constitutes the bulk of malaria cases in large parts of Asia and South America as well as recent case reports in Africa. The widespread prevalence of vivax is a challenge to global malaria elimination programmes. Vivax malaria control is particularly challenged by existence of dormant liver stage forms that are difficult to treat and are responsible for multiple relapses, growing drug resistance to the asexual blood stages and host-genetic factors that preclude use of specific drugs like primaquine capable of targeting Plasmodium vivax liver stages. Despite an obligatory liver-stage in the Plasmodium life cycle, both the difficulty in obtaining P. vivax sporozoites and the limited availability of robust host cell models permissive to P. vivax infection are responsible for the limited knowledge of hypnozoite formation biology and relapse mechanisms, as well as the limited capability to do drug screening. Although India accounts for about half of vivax malaria cases world-wide, very little is known about the vivax liver stage forms in the context of Indian clinical isolates.MethodsTo address this, methods were established to obtain infective P. vivax sporozoites from an endemic region in India and multiple assay platforms set up to detect and characterize vivax liver stage forms. Different hepatoma cell lines, including the widely used HCO4 cells, primary human hepatocytes as well as hepatocytes obtained from iPSC’s generated from vivax patients and healthy donors were tested for infectivity with P. vivax sporozoites.ResultsBoth large and small forms of vivax liver stage are detected in these assays, although the infectivity obtained in these platforms are low.ConclusionsThis study provides a proof of concept for detecting liver stage P. vivax and provide the first characterization of P. vivax liver stage forms from an endemic region in India.

Highlights

Vivax malaria is associated with significant morbidity and economic loss, and constitutes the bulk of malaria cases in large parts of Asia and South America as well as recent case reports in Africa
Malaria related deaths have showed a decline over the past decade, owing to the reduction in the number of Plasmodium falciparum malaria cases, which is mainly due to the fact that the national malaria control programmes in the past have always mainly focused on the most pathogenic and virulent form, P. falciparum [2] and the predominance of falciparum malaria within the African continent
We report the establishment of a reproducible method for generation of P. vivax sporozoites from Indian clinical isolates, infection conditions in multiple platforms, including different hepatoma cell lines, primary human hepatocytes and vivax patient derived induced pluripotent stem cells (iPSCs) generated hepatocytes

Summary

Introduction

Vivax malaria is associated with significant morbidity and economic loss, and constitutes the bulk of malaria cases in large parts of Asia and South America as well as recent case reports in Africa. The Global Technical Strategy for Malaria 2016–2030 has set major ambitious targets for malaria eradication, namely, the elimination of malaria from at least 35 countries and reduction of malaria incidence and mortality by at least 90% by 2030 [1] Such global efforts are constantly challenged by relapsing malaria species and the risk of expanding drug resistance. Despite such challenges, malaria related deaths have showed a decline over the past decade, owing to the reduction in the number of Plasmodium falciparum malaria cases, which is mainly due to the fact that the national malaria control programmes in the past have always mainly focused on the most pathogenic and virulent form, P. falciparum [2] and the predominance of falciparum malaria within the African continent. There is an urgent need for development of new class of drugs acting on vivax liver stages [8]

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