Background
The availability of field-deployable malaria rapid diagnostic tests (RDTs) in recent years has markedly facilitated access to malaria diagnostics. Since World Health Organization (WHO) recommendations in 2010 to test all suspected malaria cases [
1]. RDTs have gained a crucial role in the management of clinical episodes, as well as for malaria surveillance. Malaria RDTs have supplanted conventional light microscopy in many endemic areas as standard practice, accounting in 2017 for 75% of all diagnostic tests done in sub-Saharan Africa, where most RDTs are distributed (66%) [
2]. The vast majority of RDTs used worldwide are based on the detection of parasite bioproduct histidine-rich protein 2 (PfHRP2), expressed only in
Plasmodium falciparum, and the parasite metabolic enzyme lactate dehydrogenase (pLDH), present in all human-infecting
Plasmodium species.
PfHRP2 is a water-soluble glycoprotein produced by
P. falciparum throughout its asexual lifecycle and early sexual stages; it is expressed on the surface of infected erythrocytes and released into the peripheral blood circulation during schizogony [
3,
4]. Given the ability of mature
P. falciparum parasites to sequester in vascular beds during the last half of their asexual life-cycle, where they are not accessible for microscopic diagnosis, it has been proposed that the quantitative detection of PfHRP2 can provide a more accurate measurement of parasite biomass and potentially assist in determining the prognosis of severe malaria [
5‐
7]. During pregnancy,
P. falciparum infections can remain undetectable in peripheral blood as the parasites sequester in the intervillous spaces of the placenta by specific adhesion to chondroitin sulfate A [
8,
9]. In such scenario, PfHRP2-detecting RDTs have been shown to have higher sensitivity on peripheral blood compared to conventional light microscopy [
10], although still lower than PCR [
11].
RDTs detecting PfHRP2 only are the most widely used products [
12], accounting for 66% of the 276 million RDTs sold worldwide in 2017 [
2]. Nonetheless it has been suggested that PfHRP2-detecting RDTs have limited clinical specificity for diagnosis of current malaria infection in areas of high transmission [
13] and following treatment [
14,
15] due to the persistence of the protein in the blood circulation after parasite clearance. The time span of a positive test result following parasite clearance is mainly dependent on the duration and density of parasitaemia prior to treatment, with values ranging from 26 days in Ugandan children with parasitaemia less than 1000 parasites per microlitre (p/μl) up to 37 days for parasite density > 50,000 p/μl [
16].
The parasite LDH is a metabolic enzyme required for survival and is produced by all five
Plasmodium species infective to humans [
17,
18]. In contrast to PfHRP2, pLDH does not persist in blood after clearance of malaria infections and is therefore a better marker of acute and current infection [
19]. Upon treatment, pLDH clearance in blood has been shown to closely track with that of parasites, suggesting pLDH to be a suitable predictor for treatment failure [
20]. However, sensitivity of RDTs based on this antigen is generally lower than that of PfHRP2-based RDTs [
21].
Currently, enzyme-linked immunoabsorbent assay (ELISA) is the standard practice immunoassay for the detection and quantification of PfHRP2 and pLDH, and is used as an external validation tool for RDTs performance. 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‐
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. In this work, a high-throughput quantitative suspension array approach based on the Luminex technology that allows for the simultaneous and highly sensitive detection and quantification of PfHRP2 and pLDH antigens in different biological samples (whole blood, plasma and dried blood spots) collected from individuals living in malaria-endemic regions is presented. This assay provides an additional tool to externally evaluate the performance of new generation antigen-detecting malaria RDTs, and can be used for research purposes to address biological questions such as PfHRP2 persistence and the relationship between antigen levels and disease severity.
Discussion
In the present study, 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 is described. The qSAT allows the determination of 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,
37]. 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.
The performance of the bead suspension array to quantify PfHRP2 and pLDH was evaluated using reference recombinant proteins as well as cultured parasites, and in different biofluids from malaria-exposed and malaria-naïve individuals. The assay is selective for the target antigens and has an analytical range of 6.8–762.8 and of 78.1–17,076.6 pg/ml for PfHRP2 and
P. falciparum pLDH, respectively. Additionally, the assay can also quantify
P. vivax pLDH down to 1211.6 pg/ml. The assay analytical sensitivity to detect PfHRP2 is comparable to that of a recently developed bead suspension assay based on Luminex technology [
25], as well as to other immunoassays that use different technologies [
20,
27]. This suggests that with the current technology available for the quantification of PfHRP2 using antibodies, the lowest limit of detection achievable is in the range of 0.5–10 pg/ml. The limit of detection for pLDH is more divergent across assays, ranging from approximately 10 pg/ml [
27] up to 4000 pg/ml [
25], but in all assays it is always higher than that for PfHRP2. This underpins the need to further improve the sensitivity of pLDH-based diagnostics.
The bead suspension array described here can successfully be used as for detection and quantification of PfHRP2 and pLDH in whole blood, eluted DBS and plasma or serum samples. The concentration of eluted PfHRP2 from DBS was equivalent to approximately a 1:20 dilution from whole blood, similarly to previously reported data [
38]. Differently, for pLDH it was found that antigen concentration in eluted DBS corresponds to a 1:60 whole blood dilution, which differs from previously published data showing no differences in antigen recovery between PfHRP2 and pLDH [
20]. However, such differences could be explained by the different extraction methodologies and storage conditions used.
The quantification of PfHRP2 and pDLH is performed by interpolating MFI values to a regression curve fitted from a calibration curve consisting of recombinant proteins PfHRP2 type A and
P. falciparum pLDH. Particularly for PfHRP2, the use of a single recombinant protein as a reference material to quantify antigen levels in field samples may provide an approximate estimate of the true concentration. PfHRP2 contains sequences rich in histidine that form the epitopes targeted by the mAbs in RDTs [
39], which have been shown to be highly polymorphic in sequence composition of the repeated motifs, as well as in overall length and number of repeated motifs between different parasite strains [
39]. Baker
et al. classified PfHRP2 as types A, B, or C depending on the frequency of two epitope repeats (named type 2 and type 7) which confer increased reactivity to mAbs in RDTs [
39,
40]. According to this classification, PfHRP2 Type A comprises the higher number of repeat types 2 and 7, followed by PfHRP2 Type B, and finally PfHRP2 Type C. These results on the detection of different PfHRP2 types (see Additional file
2: Fig. S1A) align with these data and resemble recently published results [
24,
25].
An overall positive, significant correlation between antigen levels and parasite densities similar to that found in previous studies was observed [
24], although the correlation was different among the groups of samples analysed (see Additional file
3: Table S1), probably because of the type of sample used for antigen quantification, operational variations and sample storage. Of note, pLDH better correlated with parasite densities compared to PfHRP2. This finding can be explained by the fact that PfHRP2, differently from pLDH, is secreted to the blood stream and persists in circulation for several days. In addition, it was observed that the correlation between PfHRP2 and parasite densities was lower in samples from Peru compared to samples from Nigeria, whereas pLDH levels correlated very similarly to parasite densities in both groups of samples. The high number of suspected
P. falciparum-positive samples with
pfhrp2 gene deletions within the group of samples from Peru most probably explains this finding.
A potential limitation of the current assay is that it was not evaluated for possible cross-reactivity of anti-PfHRP2 mAbs with PfHRP3, a
P. falciparum protein homologous to PfHRP2 [
41], which is thought to contribute to the detection sensitivity of PfHRP2-based RDTs [
42,
43]. Another limitation of PfHRP2-detecting immunoassays, including RDTs, is the recent global spread of
P. falciparum populations lacking the
pfhrp2 or
pfhrp3 or both genes, which lead to PfHRP2-based RDT false-negative results [
44,
45]. In this regard, the assay presented here could be used to estimate the prevalence of
P. falciparum parasites with
pfhrp2/3 deletions, although mixed infections with wild type and mutant parasites would still produce PfHRP2.
Acknowledgements
We thank the study participants; the staff of the Hospitals, clinical officers, field supervisors and data managers. We acknowledge the teams at CISM in Mozambique, at University of Antioquia in Colombia, at University Cheikh Anta Diop in Senegal, at Universidad Peruana Cayetano Heredia in Peru, and at University of Lagos in Nigeria, who conducted the recruitment of participants and sample processing. We would also like to thank Iveth González, Aida Valmaseda, Marta Vidal and Himanshu Gupta for providing important inputs for optimization of the quantitative bead suspension array; and Laura Puyol, Diana Barrios and Pau Cisteró for providing logistic support. The CISM is supported by the Government of Mozambique and the Spanish Agency for International Development (AECID). ISGlobal is a member of the CERCA Programme, Generalitat de Catalunya.
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