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Measuring the Plasmodium falciparum HRP2 protein in blood from artesunate-treated malaria patients predicts post-artesunate delayed hemolysis

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Science Translational Medicine  05 Jul 2017:
Vol. 9, Issue 397, eaaf9377
DOI: 10.1126/scitranslmed.aaf9377
  • Fig. 1. Persistence of P. falciparum HRP2 in the circulation of patients treated with artesunate or quinine.

    (A) Concentrations of P. falciparum HRP2 measured by enzyme-linked immunosorbent assay (ELISA) in whole blood from 95 patients with P. falciparum infection in Bangladesh treated with intravenous artesunate (AS). Blood samples were collected on admission at day 0 (D0) and 3 days after the start of artesunate treatment (D3). (B) Concentrations of P. falciparum HRP2 measured by ELISA in whole blood and plasma from French travelers treated for severe P. falciparum malaria with artesunate (n = 53) or quinine (n = 49). Blood samples were collected on admission at day 0 and at day 3 after the start of artesunate treatment.

  • Fig. 2. HRP2 in P. falciparum–infected erythrocytes.

    (A) Reference erythrocyte membrane immunofluorescence did not indicate HRP2 expression in asynchronous culture of human erythrocytes infected with P. falciparum strains resa 1-WT and resa 1-KO. Infected erythrocytes in culture were stained with either a mouse monoclonal anti–P. falciparum HRP2 antibody (HRP2-mAb), a mouse monoclonal anti-RESA antibody (RESA-mAb), or a human anti-RESA antibody (RESA-Ab), followed by an Alexa Fluor 488–conjugated goat anti-human immunoglobulin G (IgG). (B) HRP2 was present in all parasite blood stages. After fixation and permeabilization, cultures of RBCs infected with asexual ring to schizont stages of P. falciparum strains resa 1-WT or resa 1-KO, or gametocytes (stage V) of the NF54 strain were stained with Hoechst (for DNA) and HRP2 antibody or RESA polyclonal antibody. Merge 1, without differential interference contrast (DIC); merge 2, with DIC. (C) HRP2 was present in membranous and cytosolic extracts from P. falciparum ring–infected erythrocytes. Immunoblots using three extraction methods (SDS, Triton, and hypotonic saline) indicating HRP2 and RESA expression in RBCs infected with either resa 1-WT or resa 1-KO P. falciparum strains are shown. RBCs were lysed sequentially either with Triton and SDS buffer to analyze the cytosolic or membrane-associated proteins or with a hypotonic 5 mM sodium phosphate buffer (pH 7.4) to analyze proteins associated with RBC membranes or ghosts. Proteins were detected using anti-HRP2 or anti-RESA monoclonal antibodies.

  • Fig. 3. HRP2 persists at the plasma membrane and around the cytoplasmic vesicles of once-infected RBCs.

    (A) RESA and HRP2 parasite proteins were present in both ring-infected and once-infected (pitted) RBCs. Uninfected RBCs (uRBC) from healthy donors, samples containing ring-infected RBCs from patients at admission on day 0 before treatment with artesunate, and samples containing once-infected RBCs collected 3 days after initiation of artesunate treatment are shown. Day 0 and day 3 samples were stained with Hoechst (for DNA) and HRP2 antibody or RESA polyclonal antibody and then were analyzed by fluorescence microscopy. Data were generated using samples from five patients. (B) HRP2 was localized at the plasma membrane and around the intracytoplasmic vesicles of once-infected RBCs. Transmission electron microscopy revealed RESA (25-μm beads) and P. falciparum HRP2 (10-μm beads) proteins in ring-infected RBCs (i) and once-infected RBCs (ii to vi). HRP2 and RESA proteins were observed at the membrane-cytoskeletal complex of once-infected RBCs (ii to v), whereas only HRP2 was observed around small vesicles in the cytosol (iv to vi). Data were generated using samples from five patients. (C) P. falciparum HRP2 was present in membranous and cytosolic extracts of once-infected RBCs. Immunoblots of blood samples collected during follow-up of artesunate-treated patients with P. falciparum malaria are shown. RBCs were lysed sequentially either with Triton and SDS buffer to analyze the cytosolic or membrane-associated proteins or with a hypotonic 5 mM sodium phosphate buffer (pH 7.4) to analyze proteins associated with RBC membranes or ghosts. Proteins were detected using anti-HRP2 or anti-RESA monoclonal antibodies. Bands were quantified using the ImageJ software. Corresponding RBC intensity (set at 1) was used as a reference panel within each experiment. Data were generated using samples from five patients.

  • Fig. 4. Predicting PADH by measuring HRP2 using a rapid diagnostic dipstick test.

    (A) P. falciparum HRP2–based flow cytometry accurately quantified once-infected RBCs. Blood samples were collected from patients with severe P. falciparum malaria at admission on day 0 or 2 to 4 days after initiation of treatment with artesunate (D3). Samples were labeled after membrane permeabilization (24) using either anti-RESA polyclonal antibody (i) or anti-HRP2 monoclonal antibody (ii) or both (iii) and then were analyzed by flow cytometry. The percentage of positive cells is indicated in each quadrant. Representative data from one patient sample (22 patients were analyzed). (iv) Correlation between P. falciparum HRP2–based and RESA-based quantification using day 0 and day 3 samples from 11 patients with severe malaria treated with artesunate (22 samples in total). (B) ROC curves for the prediction of PADH using titration of P. falciparum HRP2 with an HRP2-based rapid diagnostic test at admission on day 0 and upon follow-up on day 3 (±1) or day 7 (±2) after initiation of artesunate treatment. Numbers correspond to estimated AUC. (C) Results of P. falciparum HRP2 titration using a rapid diagnostic dipstick test at admission on day 0 and upon follow-up at day 3 (±1) or day 7 (±2) after initiation of artesunate treatment. Blood samples were collected from 49 patients treated with quinine and 52 patients treated with artesunate on day 0, 3 (±1), or 7 (±2).

  • Table 1. Demographic, clinical, and parasitological characteristics of malaria patient cohorts.

    Median and range. AS, artesunate; QN, quinine; Hb, hemoglobin; Ht, hematocrit.

    Bangladesh ASFrance ASFrance QN
    n955349
    Sex ratio M/F (%)49.05:50.9546.8:52.2
    Proportion of severe
    cases (%)
    54.74100100
    Age (years)284445
    (13–70)(2–86)(4–79)
    Parasites at day 0 (%)0.696.84.95
    (0.01–23.72)(0.04–38)(0.1–28.7)
    Proportion still parasitemic
    at day 3 (%)
    15.75.427.66
    Pitting at day 3 (%)4.40.8
    (0–20.2)(0–15)
    Hb at day 0 (g/dl)12.0512.7
    (5.9–17.5)(4.8–18.7)
    Ht at day 0 (%)3134.2
    (10–89)(17.2–47)

Supplementary Materials

  • Supplementary Material for:

    Measuring the Plasmodium falciparum HRP2 protein in blood from artesunate-treated malaria patients predicts post-artesunate delayed hemolysis

    Papa Alioune Ndour,* Sébastien Larréché, Oussama Mouri, Nicolas Argy, Frédérick Gay, Camille Roussel, Stéphane Jauréguiberry, Claire Perillaud, Dominique Langui, Sylvestre Biligui, Nathalie Chartrel, Audrey Mérens, Eric Kendjo, Aniruddha Ghose, Md. Mahtab Uddin Hassan, Md. Amir Hossain, Hugh W. F. Kingston, Katherine Plewes, Arjen M. Dondorp, Martin Danis, Sandrine Houzé, Serge Bonnefoy, Marc Thellier, Charles J. Woodrow, Pierre A. Buffet,* French Artesunate Working Group

    *Corresponding author. Email: pabuffet{at}gmail.com (P.A.B.); ndourmail{at}yahoo.fr (P.A.N.)

    Published 5 July 2017, Sci. Transl. Med. 9, eaaf9377 (2017)
    DOI: 10.1126/scitranslmed.aaf9377

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    • Fig. S1. STARD flowchart of malaria patient study.

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