Diagnostic performance of a 5-plex malaria immunoassay in regions co-endemic for Plasmodium falciparum, P. vivax, P. knowlesi, P. malariae and P. ovale

Abstract

Commercial point-of-care tests remain insufficient for accurately detecting and differentiating low-level malaria infections in regions co-endemic with multiple non-falciparum species, including zoonotic Plasmodium knowlesi (Pk). A 5-plex chemiluminescent assay simultaneously measures pan-Plasmodium lactate dehydrogenase (pLDH), P. falciparum (Pf)-LDH, P. vivax (Pv)-LDH, Pf-histidine-rich protein-2 (HRP2), and C-reactive protein. We assessed its diagnostic performance on whole blood (WB) samples from 102 healthy controls and 306 PCR-confirmed clinical cases of Pf, Pv, Pk, P. malariae (Pm) and P. ovale (Po) mono-infections from Southeast-Asia. We confirm its excellent HRP2-based detection of Pf. Cross-reactivity of Pf-LDH with all non-falciparum species tested was observed (specificity 57.3%). Pv-LDH performance was suboptimal for Pv (93.9% sensitivity and 73.9% specificity). Poor specificity was driven by strong Pk cross-reactivity, with Pv-LDH detecting 93.9% of Pk infections. The pan-LDH-to-Pf-LDH ratio was capable of discerning Pv from Pk, and robustly differentiated Pf from Pm or Po infection, useful in regions with hrp2/3 deletions. We tested the platform’s performance in plasma for the first time, with WB outperforming plasma for all analytes except Pv-LDH for Pk. The platform is a promising tool for WB malaria diagnosis, although further development is warranted to improve its utility in regions co-endemic for multiple non-falciparum species.

Diagnostic accuracy and limit of detection of ten malaria parasite lactate dehydrogenase-based rapid tests for Plasmodium knowlesi and P. falciparum

“…In the current study, P. knowlesi parasites from both clinical and laboratory-cultured samples were also detected by all pan-pLDH and Pv-pLDH components tested, meaning they are likely unable to differentiate P. vivax. The quantified ratio of pan-to Pv-pLDH responses could provide additional information if this approach was used during the development of the RDTs (Kho et al, 2022), however this requires further investigation. With the exception of CareStart ™ PAN, in vitro LoD values from the pan-pLDH based RDTs were generally higher than the lowest detected parasitaemia of the clinical samples.…”

Diagnostic accuracy and limit of detection of ten malaria parasite lactate dehydrogenase-based rapid tests for Plasmodium knowlesi and P. falciparum

“…In the current study, P. knowlesi parasites from both clinical and laboratory-cultured samples were also detected by all pan-pLDH and Pv -pLDH components tested, meaning they are likely unable to differentiate P. vivax . The quantified ratio of pan-to Pv -pLDH responses could provide additional information if this approach was used during the development of the RDTs (51), however this requires further investigation. With the exception of CareStart™ PAN, in vitro LoD values from the pan-pLDH based RDTs were generally higher than the lowest detected parasitaemia of the clinical samples.…”

Characterisation ofPlasmodium vivaxlactate dehydrogenase dynamics inP. vivaxinfections

“…Quansys 5-plex ELISA PvLDH levels in VIS were measured in whole blood samples collected between day 4 and day of treatment, and every 12 hours after treatment until 120 hours. PvLDH levels in the samples above were measured using the Q-Plex™ Human Malaria assay (Quansys Biosciences, Logan, UT, USA) as previously described 26 . The Q-View™ Imager Pro was used to capture chemiluminescent images of each plate which was then quantitatively analysed using Q-View™ software (Quansys Biosciences).…”

“…Using this relationship, we transformed the VIS PvLDH data to calibrate with the ex vivo data measured by inhouse ELISA. The relationship between inhouse ELISA and Quansys PvLDH concentrations was established using a retrospective cross-sectional dataset of 58 vivax malaria patients in Sabah, Malaysia, who had whole blood PvLDH concentrations measured by both inhouse ELISA and Quansys methods26 . Using a Bayesian approach, we fitted a linear model to the logtransformed ELISA and Quansys data from Malaysian patients (Fig.S6in the Supplementary Information).…”