Measures of malaria burden using microscopy and rapid diagnostic tests (RDTs) in cross-sectional household surveys may incompletely describe the burden of malaria in low-transmission settings. This study describes the pattern of malaria transmission in Ethiopia using serological antibody estimates derived from a nationwide household survey completed in 2015.

Multiplex assays for malaria antigen detection can gather data from large sample sets, but considerations for the consistency and quality assurance (QA) of mass testing lack evaluation. We present a QA framework for a study occurring November 2019 to March 2020 involving 504 assay plates detecting four Plasmodium antigens: pan-Plasmodium aldolase and lactate dehydrogenase (LDH), histidine-rich protein 2 (HRP2), P. vivax LDH (PvLDH). Controls on each plate included buffer blank, antigen negative blood, and 4-point positive dilution curve. The blank and negative blood provided consistently low signal for all targets except for pAldolase, which showed variability. Positive curve signals decreased throughout the 5-month study duration but retained a coefficient of variation (CV) of < 5%, with the exception of HRP2 in month 5 (CV of 11%). Regression fittings for inter-plate control signals provided mean and standard deviations (SDs), and of 504 assay plates, 6 (1.2%) violated the acceptable deviation limits and were repeated. For the 40,272 human blood samples assayed in this study, of 161,088 potential data points (each sample × 4 antigens), 160,641 (99.7%) successfully passed quality checks. The QA framework presented here can be utilized to ensure quality of laboratory antigen detection for large sample sets.

The majority of Plasmodium falciparum malaria diagnoses in Africa are made using rapid diagnostic tests (RDTs) that detect histidine-rich protein 2. Increasing reports of false-negative RDT results due to parasites with deletions of the pfhrp2 and/or pfhrp3 genes (pfhrp2/3) raise concern about existing malaria diagnostic strategies. We previously identified pfhrp2-negative parasites among asymptomatic children in the Democratic Republic of the Congo (DRC), but their impact on diagnosis of symptomatic malaria is unknown. We performed a cross-sectional study of false-negative RDTs in symptomatic subjects in 2017. Parasites were characterized by microscopy; RDT; pfhrp2/3 genotyping and species-specific PCR assays; a bead-based immunoassay for Plasmodium antigens; and/or whole-genome sequencing. Among 3627 symptomatic subjects, 427 (11.8%) had RDT-/microscopy + results. Parasites from eight (0.2%) samples were initially classified as putative pfhrp2/3 deletions by PCR, but antigen testing and whole-genome sequencing confirmed the presence of intact genes. 56.8% of subjects had PCR-confirmed malaria. Non-falciparum co-infection with P. falciparum was common (13.2%). Agreement between PCR and HRP2-based RDTs was satisfactory (Cohen's kappa = 0.66) and superior to microscopy (0.33). Symptomatic malaria due to pfhrp2/3-deleted P. falciparum was not observed. Ongoing HRP2-based RDT use is appropriate for the detection of falciparum malaria in the DRC.

Asymptomatic malaria infections can serve as potential reservoirs for malaria transmission. The density of parasites contained in these infections range from microscopic to submicroscopic densities, making the accurate detection of asymptomatic parasite carriage highly dependent on the sensitivity of the tools used for the diagnosis. This study sought to evaluate the sensitivities of a variety of molecular and serological diagnostic tools at determining the prevalence of asymptomatic Plasmodium falciparum parasite infections in two communities with varying malaria parasite prevalence.