Evidence for histidine-rich protein 2 immune complex formation in symptomatic patients in Southern Zambia

Background: Rapid diagnostic tests based on histidine-rich protein 2 (HRP2) detection are the primary tools used to detect Plasmodium falciparum malaria infections. Recent conflicting reports call into question whether alpha-HRP2 antibodies are present in human host circulation and if resulting immune complexes could interfere with HRP2 detection on malaria RDTs. This study sought to determine the prevalence of immune-complexed HRP2 in a low-transmission region of Southern Zambia. Methods: An ELISA was used to quantify HRP2 in patient sample DBS extracts before and after heat-based immune complex dissociation. A pull-down assay reliant on proteins A, G, and L was developed and applied for IgG and IgM capture and subsequent immunoprecipitation of any HRP2 present in immune complexed form. A total of 104 patient samples were evaluated using both methods. Results: Immune-complexed HRP2 was detectable in 17% (18/104) of all samples evaluated and 70% (16/23) of HRP2-positive samples. A majority of the patients with samples containing immune-complexed HRP2 had P. falciparum infections (11/18) and were also positive for free HRP2 (16/18). For 72% (13/18) of patients with immune-complexed HRP2, less than 10% of the total HRP2 present was in immune-complexed form. For the remaining samples, a large proportion (>= 20%) of total HRP2 was complexed with alpha-HRP2 antibodies. Conclusions: Endogenous alpha-HRP2 antibodies form immune complexes with HRP2 in the symptomatic patient population of a low-transmission area in rural Southern Zambia. For the majority of patients, the percentage of HRP2 in immune complexes is low and does not affect HRP2-based malaria diagnosis. However, for some patients, a significant portion of the total HRP2 was in immune-complexed form. Future studies investigating the prevalence and proportion of immune-complexed HRP2 in asymptomatic individuals with low HRP2 levels will be required to assess whether alpha-HRP2 antibodies affect HRP2 detection for this portion of the transmission reservoir.