Malaria is a vector-borne disease of major public health relevance worldwide. In 2015, the World Health Organization (WHO) reported 212 million new cases and 429,000 deaths, most of which were attributed to
Plasmodium falciparum [
1]. Significant reductions, however, occurred from 2010 to 2015, leading to 21% and 29% decreases in incidence and mortality, respectively [
1]. These improvements in disease burden have been largely attributed to vector control, and improved and accessible diagnostics and treatment [
1]. As a result, at least 21 countries are positioned for elimination with many others following suit [
1,
2], but in order to maintain such progress, continued commitment to malaria control strategies will be required. In particular, the role of current diagnostics has become an increasingly important issue as low density infections have been identified at high rates in low prevalence settings [
2,
3]. These low density parasite infections serve as reservoirs and are predicted to make up 20–50% of human-to-mosquito transmission [
4]. While RDTs and microscopy are considered the current diagnostic standards for malaria, the limit of detection (LoD) for each tool, 5 parasites/µL in expert reference laboratories and 20 parasites/µL more generally for blood film microscopy; 100–200 parasites/µL and 800 picograms (pg)/mL histidine-rich protein 2 (HRP2) for
P. falciparum RDTs, is not sufficient for detecting low density infections [
4‐
10]. Highly sensitive and specific field deployable diagnostic tools to detect low density infections may provide more accurate estimates of ongoing malaria transmission as well as render case detection-based elimination strategies more effective. The Alere™ ultrasensitive
P. falciparum HRP2-based RDT with a greater than tenfold improvement in limit-of-detection for HRP2 over previously available RDTs was launched in April 2017 [
11,
12]. The development and performance of these tools for low density infections also require complementary laboratory-based reference assays for the same analytes that can confirm performance of these tests.
Currently available standard ELISAs for HRP2 do not attain low enough LoDs for HRP2 to serve as useful reference assays for new highly sensitive HRP2-based RDTs or to define HRP2 distributions in populations with a large proportion of low density infections [
13,
14]. While suitably sensitive assays for HRP2 already exist, the platforms are not readily available to laboratories outside the research context [
11,
15]. A standard ELISA remains the most accessible platform to most laboratories. A novel highly sensitive Alere™ Malaria Ag
Pf ELISA (HS ELISA) has been developed. The HS ELISA has a similar platform and protocol compared to current commercial
P. falciparum HRP2-based ELISA kits, but requires a smaller volume of blood, 50 μL versus 100 μL, respectively, making it an attractive reference tool for large-scale use in field laboratories. In this study, HS ELISA performance against
P. falciparum HRP2 was characterized using
P. falciparum recombinant HRP2, a panel of
P. falciparum native culture specimens, and clinical whole blood specimens from Myanmar and Uganda.