A new highly sensitive enzyme-linked immunosorbent assay for the detection of Plasmodium falciparum histidine-rich protein 2 in whole blood

Abstract

BackgroundThe detection of submicroscopic infections in low prevalence settings has become an increasingly important challenge for malaria elimination strategies. The current field rapid diagnostic tests (RDTs) for Plasmodium falciparum malaria are inadequate to detect low-density infections. Therefore, there is a need to develop more sensitive field diagnostic tools. In parallel, a highly sensitive laboratory reference assay will be essential to evaluate new diagnostic tools. Recently, the highly sensitive Alere™ Malaria Ag P.f ELISA (HS ELISA) was developed to detect P. falciparum histidine-rich protein 2 (HRP2) in clinical whole blood specimens. In this study, the analytical and clinical performance of the HS ELISA was determined using recombinant P. falciparum HRP2, P. falciparum native culture parasites, and archived highly pedigreed clinical whole blood specimens from Karen village, Myanmar and Nagongera, Uganda.ResultsThe HS ELISA has an analytical sensitivity of less than 25 pg/mL and shows strong specificity for P. falciparum HRP2 when tested against P. falciparum native culture strains with pfhrp2 and pfhrp3 gene deletions. Additionally, the Z′-factor statistic of 0.862 indicates the HS ELISA as an excellent, reproducible assay, and the coefficients of variation for inter- and intra-plate testing, 11.76% and 2.51%, were acceptable. Against clinical whole blood specimens with concordant microscopic and PCR results, the HS ELISA showed 100% (95% CI 96.4–100) diagnostic sensitivity and 97.9% (95% CI 94.8–99.4) diagnostic specificity. For P. falciparum positive specimens with HRP2 concentrations below 400 pg/mL, the sensitivity and specificity were 100% (95% CI 88.4–100) and 88.9% (95% CI 70.8–97.6), respectively. The overall sensitivity and specificity for all 352 samples were 100% (CI 95% 96–100%) and 97.3% (CI 95% 94–99%).ConclusionsThe HS ELISA is a robust and reproducible assay. The findings suggest that the HS ELISA may be a useful tool as an affordable reference assay for new ultra-sensitive HRP2-based RDTs.

Systematic review of the status of pfhrp2 and pfhrp3 gene deletion, approaches and methods used for its estimation and reporting in Plasmodium falciparum populations in Africa: review of published studies 2010–2019

“…Studies have shown the failure of HRP2 based and the ability of non-HRP2 RDTs to detect pfhrp2/3 gene-deleted parasites in P. falciparum infected samples [6, 11, 12]. Indeed, the advance in the development of robust diagnostic tools to detect gene-deleted parasites is enormous [4, 21, 25, 32–35, 38, 39]. In addition to the current molecular and serological tools, Plucinski et al have developed a bead-based multiplex assay that simultaneously detects parasite aldolase, parasite lactate dehydrogenase and histidine rich protein 2 increasing the possibility of detecting gene-deleted parasites [20].…”

Quantification of malaria antigens PfHRP2 and pLDH by quantitative suspension array technology in whole blood, dried blood spot and plasma

“…ELISAs are however costly, time and sample consuming, and generally only allow for the detection of one analyte at the time. The recent release of a highly-sensitive RDT for PfHRP2 (Alere ™ Malaria Ag P.f ), with two to ten-fold higher sensitivity than other currently available RDTs [22,23], as well as the work in progress to develop new generation pLDH-based RDTs, underpins the need for new highly-sensitive, laboratory-based, reference immunoassays than can provide lower limit of detection than classical ELISAs [24][25][26][27]. Highly sensitive quantitative assays should not only be a more suitable tool for validation of new-generation RDTs, but could also be used to better understand antigen kinetics, particularly that of PfHRP2, and to support malaria surveillance.…”

Quantification of malaria antigens PfHRP2 and pLDH by quantitative suspension array technology in whole blood, dried blood spot and plasma

“…In the present study, we have established a quantitative suspension array based on Luminex technology for the simultaneous detection and quantification of P. falciparum HRP2 and P. falciparum and P. vivax pLDH, which allows to determine protein concentrations as low as 6.0, 56.1 and 1042.7 pg/ml, respectively. Hence, the assay provides increased sensitivity compared to commercially available ELISA kits which have LODs of approximately 400 pg/ml and 1000 pg/ml for PfHRP2 and pLDH, respectively (27,39). The assay shows good levels of dilution linearity, accuracy and precision, and can be used to effectively and rapidly quantify malaria antigens in large quantities of different biosamples.…”