ABSTRACT
Background Malaria diagnostics by rapid diagnostic tests (RDTs) relies primarily on the qualitative detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and Plasmodium sp lactate dehydrogenase (pLDH). As novel RDTs with increased sensitivity are being developed and implemented as point of care diagnostics, highly sensitive laboratory based assays are needed for evaluating RDTs performance. Here, a quantitative suspension array technology (qSAT) was developed, validated and applied for the simultaneous detection of PfHRP2 and pLDH in a variety of clinical samples (whole blood, plasma and dried blood spots) from different endemic countries.
Results The qSAT was specific for the target antigens, with analytical ranges of 6.8 to 762.8 pg/ml for PfHRP2 and 78.1 to 17076.6 pg/ml for P. falciparum (Pf-LDH). The assay detected P. vivax LDH (Pv-LDH) at a lower sensitivity than Pf-LDH (analytical range of 1093.20 to 187288.5 pg/ml). Both PfHRP2 and pLDH levels determined using the qSAT showed to positively correlate with parasite densities determined by quantitative PCR (Spearman r=0.59 and 0.75, respectively) as well as microscopy (Spearman r=0.40 and 0.75, respectively), suggesting the assay to be a good predictor of parasite density.
Conclusion This immunoassay can be used as a reference test for the detection and quantification of PfHRP2 and pLDH, and could serve for external validation of RDTs performance, to determine antigen persistence after parasite clearance, as well as a complementary tool to assess malaria burden in endemic settings.
Footnotes
Author email addresses: Xavier Martiáñez-Vendrell (xavier.martianez{at}isglobal.org); Alfons Jiménez (alfons.jimenez{at}isglobal.org); Ana Vásquez (amvc.ana{at}gmail.com); Ana Campillo (apillu{at}gmail.com); Sandra Incardona (sandra.incardona{at}finddx.org); Raquel González (raquel.gonzalez{at}isglobal.org); Dionicia Gamboa (dionigamboa{at}yahoo.com); Katherine Torres (kathjess2000{at}yahoo.com), Wellington Oyibo (woyibo{at}unilag.edu.ng); Babacar Faye (bfaye67{at}yahoo.fr); Eusebio Macete (eusebio.macete{at}manhica.net); Clara Manéndez (clara.menendez{at}isglobal.org); Xavier Ding (xavier.ding{at}finddx.org); and Alfredo Mayor (alfredo.mayor{at}isglobal.org).
Abbreviations
- α
- anti
- Ag
- antigen;
- CI
- confidence intervals;
- DBS
- dried blood spot;
- EDC
- 1-Ethyl-3-[3-dimethylaminopropyl] carbodimide hydrochloride;
- ELISA
- enzyme-linked immunoabsorbent assay;
- GM
- Geometric mean;
- IgG
- immunoglobulin G;
- LLOD
- lower limit of detection;
- LLOQ
- lower limit of quantification;
- LM
- light microscopy;
- mAbs
- monoclonal antibodies;
- MFI
- median fluorescence intensity;
- p/μl
- parasites per microliter;
- PCR
- polymerase chain reaction;
- Pf
- Plasmodium falciparum;
- pg/ml
- pictograms per millilitre;
- PfHRP2
- Plasmodium falciparum histidine-rich protein 2;
- PfHRP3
- Plasmodium falciparum histidine-rich protein 3;
- pLDH
- parasite lactate dehydrogenase;
- Pv
- Plasmodium vivax;
- qPCR
- quantitative polymerase chain reaction;
- qSAT
- quantitative suspension array technology;
- RDT
- rapid diagnostic test;
- RT
- room temperature;
- SD
- standard deviation;
- Sulfo-NHS
- (N-hydroxysulfosuccinimide);
- ULOQ
- upper limit of quantification;
- µl
- microliter;
- 5PL
- five parameters logistic;
- °C
- degrees Celsius;
- %CV
- percent coefficient of variation;
- %DEV
- percent deviation.