Diagnostic Characteristics of Lactate Dehydrogenase on a Multiplex Assay for Malaria Detection Including the Zoonotic Parasite Plasmodium knowlesi

Abstract

Plasmodium lactate dehydrogenase (pLDH) is a common target in malaria rapid diagnostic tests (RDTs). These commercial antibody capture assays target either Plasmodium falciparum–specific pLDH (PfLDH), P. vivax–specific pLDH (PvLDH), or a conserved epitope in all human malaria pLDH (PanLDH). However, there are no assays specifically targeting P. ovale, P. malariae or zoonotic parasites such as P. knowlesi and P. cynomolgi. A malaria multiplex array, carrying the specific antibody spots for PfLDH, PvLDH, and PanLDH has been previously developed. This study aimed to assess potential cross-reactivity between pLDH from various Plasmodium species and this array. We tested recombinant pLDH proteins, clinical samples for P. vivax, P. falciparum, P. ovale curtisi, and P. malariae; and in vitro cultured P. knowlesi and P. cynomolgi. P. ovale-specific pLDH (PoLDH) and P. malariae-specific pLDH (PmLDH) cross-reacted with the PfLDH and PanLDH spots. Plasmodium Knowlesi-specific pLDH (PkLDH) and P. cynomolgi-specific pLDH (PcLDH) cross-reacted with the PvLDH spot, but only PkLDH was recognized by the PanLDH spot. Plasmodium ovale and P. malariae can be differentiated from P. falciparum by the concentration ratios of PanLDH/PfLDH, which had mean (range) values of 4.56 (4.07–5.16) and 4.56 (3.43–6.54), respectively, whereas P. falciparum had a lower ratio of 1.12 (0.56–2.61). Plasmodium knowlesi had a similar PanLDH/PvLDH ratio value, with P. vivax having a mean value of 2.24 (1.37–2.79). The cross-reactivity pattern of pLDH can be a useful predictor to differentiate certain Plasmodium species. Cross-reactivity of the pLDH bands in RDTs requires further investigation.

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

“…Importantly, we found that the assay requires further development for precise identification of non-falciparum species. The high level conservation of the LDH protein among the different Plasmodium species, with resultant cross-reacting antibody specificity, means that the Pf-LDH cannot distinguish P. falciparum from P. malariae 7 and P. ovale , and is consistent with previous findings 10 . Despite this, it was possible to differentiate P. malariae and P. ovale from P. falciparum by using the lack of HRP2 positivity.…”

“…In addition to demonstrating known P. knowlesi cross-reactivity with Pf-LDH 17 , 19 , the platform’s diagnostic performance for P. knowlesi revealed strong cross reactivity with the P. vivax marker (Pv-LDH) as previously reported with both cultured A1H1 strain samples 10 and using other diagnostic methods 18 , 34 , 35 . The superior sensitivity of RDTs targeting Pv-LDH in detecting P. knowlesi over P. vivax has previously been reported 35 and was seen in our plasma samples.…”

“…The performance of 4-plex 8 and 5-plex 7 assays for antigen-based malaria screening has been tested on whole blood from asymptomatic and symptomatic infections with P. falciparum , P. vivax , P. malariae and P. ovale and on matching dry blood-spots 9 , reporting high sensitivity for P. falciparum , including for those with hrp2/3 deletions. Apart from P. cynomolgi 10 , genus-specific pLDH has enabled detection of all human Plasmodium species in whole blood with varying sensitivity. However, because of the high level of conservation of the Plasmodium protein, anti-pLDH antibodies designed to detect species-specific epitopes in the protein have demonstrated cross-reactivity with other species, resulting in loss of specificity 10 .…”

“…Apart from P. cynomolgi 10 , genus-specific pLDH has enabled detection of all human Plasmodium species in whole blood with varying sensitivity. However, because of the high level of conservation of the Plasmodium protein, anti-pLDH antibodies designed to detect species-specific epitopes in the protein have demonstrated cross-reactivity with other species, resulting in loss of specificity 10 .…”

“…As a zoonosis, its epidemiology and strategies for control differ significantly from other species causing malaria, making accurate diagnosis important. The 5-plex Quansys platform has significant cross-reactivity between Pv-LDH and P. knowlesi generated from in-vitro cultures 10 . Detection of low parasitemia with high specificity and sensitivity for all Plasmodium species causing human malaria in co-endemic areas is essential.…”