# In vitro cultured malaria hypnozoites leave a footprint of specific metabolites

## TL;DR

This study identifies specific chemical markers released by dormant malaria parasites in the liver, which could lead to a test for detecting hidden infections.

## Contribution

The study presents a proof-of-concept for identifying metabolite markers specific to Plasmodium hypnozoites using in vitro cultures.

## Key findings

- Specific metabolites were detected in the supernatant of hypnozoite-enriched in vitro liver stage cultures.
- These metabolites could serve as potential diagnostic markers for hypnozoite infection after further validation in animal and human models.

## Abstract

Plasmodium vivax malaria control and elimination is complicated by the presence of dormant liver stages, known as hypnozoites, that can reactivate weeks, months or years after infection giving rise to clinical and transmissible vivax malaria, without exposure to new infectious mosquito bites. Hypnozoite infection remains without symptoms and there are no diagnostic tools available to identify hypnozoite carriers. Such diagnostic tools are invaluable for precise mapping of the scale of the infection problem and for identifying individuals that would qualify for targeted drug treatment, to wipe out this hidden reservoir of malaria parasites. Targeted treatment would have considerable benefits as it would prevent the exposure of individuals without hypnozoites to the considerable side-effects of drugs such as Primaquine, which has a relatively high toxicity to people deficient in glucose-6-phosphate dehydrogenase. Here we present a Proof-of-Concept study aimed at identifying diagnostic markers for malaria hypnozoite infection, by combining in vitro Plasmodium cynomolgi hypnozoite cultures (an accessible proxy to P. vivax with nearly identical biology) with sensitive metabolomics. Specific hypnozoite-related metabolites have been identified in the supernatant of hypnozoite-enriched in vitro liver stage cultures. This suggests that, following in vivo validation of such metabolites in the P. cynomolgi/rhesus monkey model and subsequent in P. vivax-infected individuals, a rapid diagnostic test for hypnozoite infection may be developed.

Malaria caused by Plasmodium vivax is especially hard to eliminate because the parasite can hide in the liver as “sleeping” forms called hypnozoites. These dormant parasites can wake up weeks, months, or even years after the original infection and cause the disease to return—without any new mosquito bite. The problem is that people carrying these hidden parasites show no symptoms, and there are currently no tests available to detect them. This makes it difficult to know who needs treatment and leads to unnecessary drug use, which can cause serious side effects in some individuals. In this study, we explored a new way to detect hypnozoite infections by searching for chemical fingerprints—called metabolites—that are either released by these dormant parasites directly or by the liver as a result of their presence. Using a closely related malaria parasite (Plasmodium cynomolgi) grown in lab conditions, we identified specific metabolites linked to the presence of hypnozoites. This is the first step toward developing a simple test that could detect hidden malaria infections in people. If successful, such a test could improve how we treat and control P. vivax malaria, making it safer and more targeted.