Rapid Fire Presentation 8th International Conference on Plasmodium vivax Research 2022

Development of a new Plasmodium vivax LDH secondary diagnostic standard using P. knowlesi orthologue replacement (#328)

Giulia Pianta 1 , Franziska Mohring 1 , James Thomas 1 , Adela Nacer 2 , Charles Olomu 2 , Lynne Harris 2 , Paul Bowyer 2 , Robert Moon 1
  1. London School of Hygiene and Tropical Medicine, London, LONDON, United Kingdom
  2. NIBSC, Potters Bar, United Kingdom

Rapid diagnostic tests (RDTs) detecting the pan-malaria antigen lactate dehydrogenase (LDH) have become increasingly important in the diagnosis of non-falciparum infections. However, current monoclonal antibodies employed by manufacturers do not always consistently detect all malaria species. This is aggravated by the lack of appropriate WHO-defined International Standards (IS). P. falciparum is adapted for culturing in a lab setting, and this material is used to generate the WHO IS used for prequalification. However, generation of IS for other malaria species relies on time-consuming and expensive clinical sample collection, and use of recombinant proteins is not recommended. While new IS were recently developed for P. vivax, it is not sustainable for other species such as P. ovale spp, resulting in uncertain RDT detection rates for these species.

We took advantage of the adaptation of P. knowlesi to grow in vitro and the successful application of the CRISPR/Cas9 technique for orthologue replacement to substitute the pkldh gene with its orthologue from P. vivax and P. ovale spp. These lines were tested against various RDTs from different companies to compare species specific reactivity and sensitivity.

The transgenic PkPvLDHOR line was detected by all RDTs assessed. The detection intensity of our transgenic line was higher than the WHO-derived P.vivax IS from clinical isolates, even at lower dilutions. No cross-reactivity occurred with PkLDH samples, despite their evolutionary similarity. Moreover, comparisons of lines expressing PocLDH and PowLDH revealed distinct detection profiles varying between RDT devices. This provided a proof of concept for the generation of P. knowlesi transgenic lines expressing polymorphic variants of PvLDH and PoLDH.

This approach provides the basis for the creation of a panel of species-specific LDH-expressing transgenic lines constituting a renewable source of biological material to establish WHO International Standards, improve validation of existing RDTs and development of more accurate pan-LDH RDTs.