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

Christine F Markwalter,David W Wright,Lwiindi Mudenda,Mindy Leelawong,Armin Nourani,Philip E Thuma,Danielle W Kimmel,Saidon Mbambara

doi:10.1186/s12936-018-2400-8

Christine F Markwalter, David W Wright + Show 6 more

Open Access

https://doi.org/10.1186/s12936-018-2400-8

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Abstract

BackgroundRapid 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 α-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.MethodsAn 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.ResultsImmune-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 α-HRP2 antibodies.ConclusionsEndogenous α-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 α-HRP2 antibodies affect HRP2 detection for this portion of the transmission reservoir.

Highlights

Rapid diagnostic tests based on histidine-rich protein 2 (HRP2) detection are the primary tools used to detect Plasmodium falciparum malaria infections
Most rapid diagnostic test (RDT) specific for this species rely on the detection of P. falciparum histidine-rich protein 2 (HRP2), which was the first antigen targeted in commercial tests [2]
Dissociation of pre‐formed HRP2 immune complexes A series of laboratory controls were performed before analysis of patient samples

Summary

Introduction

Rapid diagnostic tests based on histidine-rich protein 2 (HRP2) detection are the primary tools used to detect Plasmodium falciparum malaria infections. The World Health Organization estimates that 312 million RDTs were delivered globally [1] These common tests are most often formatted as lateral flow assays, which use antibodies to capture and detect malarial parasite proteins. Most RDTs specific for this species rely on the detection of P. falciparum histidine-rich protein 2 (HRP2), which was the first antigen targeted in commercial tests [2]. The unique structure of HRP2 makes it an advantageous biomarker for malaria detection; multiple epitope copies on a single protein result in high-avidity interactions with antibodies present in RDTs, leading to effective capture and detection of HRP2. It is not surprising that the most sensitive RDTs on the market are based on HRP2 detection, though performance varies significantly by manufacturer [10]

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