Screening for Pfhrp2/3-Deleted Plasmodium falciparum, Non-falciparum, and Low-Density Malaria Infections by a Multiplex Antigen Assay

Abstract

<div class="section"> <a class="named-anchor" id="s1"> <!-- named anchor --> </a> <h5 class="section-title" id="d7877757e290">Background</h5> <p id="d7877757e292">Detection of <i>Plasmodium</i> antigens provides evidence of malaria infection status and is the basis for most malaria diagnosis. </p> </div><div class="section"> <a class="named-anchor" id="s2"> <!-- named anchor --> </a> <h5 class="section-title" id="d7877757e298">Methods</h5> <p id="d7877757e300">We developed a sensitive bead-based multiplex assay for laboratory use, which simultaneously detects pan- <i>Plasmodium</i> aldolase (pAldo), pan- <i>Plasmodium</i> lactate dehydrogenase (pLDH), and <i>P. falciparum</i> histidine-rich protein 2 (PfHRP2) antigens. The assay was validated against purified recombinant antigens, monospecies malaria infections, and noninfected blood samples. To test against samples collected in an endemic setting, Angolan outpatient samples (n = 1267) were assayed. </p> </div><div class="section"> <a class="named-anchor" id="s3"> <!-- named anchor --> </a> <h5 class="section-title" id="d7877757e312">Results</h5> <p id="d7877757e314">Of 466 Angolan samples positive for at least 1 antigen, the most common antigen profiles were PfHRP2+/pAldo+/pLDH+ (167, 36%), PfHRP2+/pAldo−/pLDH− (163, 35%), and PfHRP2+/pAldo+/pLDH− (129, 28%). Antigen profile was predictive of polymerase chain reaction (PCR) positivity and parasite density. Eight Angolan samples (1.7%) had no or very low PfHRP2 but were positive for 1 or both of the other antigens. PCR analysis confirmed 3 (0.6%) were <i>P. ovale</i> infections and 2 (0.4%) represented <i>P. falciparum</i> parasites lacking <i>Pfhrp2</i> and/or <i>Pfhrp3.</i> </p> </div><div class="section"> <a class="named-anchor" id="s4"> <!-- named anchor --> </a> <h5 class="section-title" id="d7877757e329">Conclusions</h5> <p id="d7877757e331">These are the first reports of <i>Pfhrp2/3</i> deletion mutants in Angola. High-throughput multiplex antigen detection can inexpensively screen for low-density <i>P. falciparum</i>, non- <i>falciparum</i>, and <i>Pfhrp2/3</i>-deleted parasites to provide population-level antigen estimates and identify specimens requiring further molecular characterization. </p> </div><p class="first" id="d7877757e346">A multiplex malaria antigen detection assay was developed to measure pan- <i>Plasmodium</i> antigens aldolase and LDH, and <i>P. falciparum</i> HRP2. Multiplex antigen detection allowed for prediction of malaria infection status, species of infection, and detection of <i>Pfhrp2/3</i>-deleted parasites. </p>