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

Abstract

Background: Detection of Plasmodium antigens provides evidence of malaria infection status and is the basis for most malaria diagnosis.

Methods: We developed a sensitive bead-based multiplex assay for laboratory use, which simultaneously detects pan-Plasmodium aldolase (pAldo), pan-Plasmodium lactate dehydrogenase (pLDH), and P. falciparum 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.

Results: 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 P. ovale infections and 2 (0.4%) represented P. falciparum parasites lacking Pfhrp2 and/or Pfhrp3.

Conclusions: These are the first reports of Pfhrp2/3 deletion mutants in Angola. High-throughput multiplex antigen detection can inexpensively screen for low-density P. falciparum, non-falciparum, and Pfhrp2/3-deleted parasites to provide population-level antigen estimates and identify specimens requiring further molecular characterization.

Figure 1.

Titration of multiplex assay signal for recombinant aldolase, lactose dehydrogenase (LDH), and histidine-rich protein 2 (HRP2) Plasmodium proteins. Recombinant forms of Plasmodium vivax aldolase, P. falciparum, and P. vivax-specific isoforms of LDH, and P. falciparum type A, B, and C HRP2s were diluted to determine the minimal concentrations that gave appreciable median fluorescence intensity minus background (MFI-bg) assay signal. Error bars show ± standard error of the mean from 3 or 4 independent runs.

Figure 2.

Quantification of Plasmodium aldolase (pAldo), Plasmodium lactate dehydrogenase (pLDH), and histidine-rich protein 2 (HRP2) from plasma of individuals with single-species Plasmodium infection. A, Median fluorescence intensity minus background (MFI-bg) signal for multiplex antigen assay from plasma of noninfected individuals (n = 73), or persons with monoinfection of P. falciparum (n = 153), P. vivax (n = 54), P. ovale (n = 23), or P. malariae (n = 9). B, Correlation of parasite density and antigen concentration by infecting species. Regression lines are shown by dashed lines in plots and regression estimates are shown in Supplementary Table 2. C, Correlation of pAldo and pLDH antigen concentrations within individual plasma samples for all 4 different types of infecting species. Dashed lines show regression lines.

Figure 3.

Relationship of survey participants’ individual antigen profile with Plasmodium falciparum infection status and parasite density. Persons presenting to health facilities in Uige and Huambo, Angola had antigen levels for Plasmodium aldolase (pAldo), Plasmodium lactate dehydrogenase (pLDH), and P. falciparum histidine-rich protein 2 (PfHRP2) estimated by the multiplex bead assay, and had P. falciparum parasite density estimated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Ratios on x axis for each antigen profile category indicate persons positive for P. falciparum nucleic acids divided by total number tested by qRT-PCR. Horizontal lines show mean parasite density for parasite-positive persons in each antigen category.

Figure 4.

Identification of Plasmodium isolates from Angola not producing P. falciparum histidine-rich protein 2 (PfHRP2). A, Two by 2 tables of antigen positivity. Dashed circles show blood samples positive for Plasmodium aldolase (pAldo) or Plasmodium lactate dehydrogenase (pLDH) but not PfHRP2. B, Scatterplots of PfHRP2 antigen levels with pAldo and pLDH. Dashed ovals show samples positive for pan-Plasmodium antigens but not PfHRP2. Solid circle shows single sample from study with high level of pAldo and pLDH but very low PfHRP2.

Figure 5.

Screening algorithm for dried blood spots collected during health facility surveys in Angola, 2016. Percentages calculated using the total number of samples positive for any Plasmodium antigen as the denominator. *Includes 1 sample with abnormally low PfHRP2 concentration (<1000 pg/mL). **Absence of PfHRP2 protein despite successful amplification of Pfhrp2 and Pfhrp3 PCR targets. ***Does not meet World Health Organization reporting guidelines due to nonamplification of single-copy Pfmsp1 and Pfmsp2 genes. Abbreviations: pAldo, Plasmodium aldolase; PCR, polymerase chain reaction; PfHRP2, Plasmodium histidine-rich protein 2; pLDH, Plasmodium lactate dehydrogenase.