Multiplex malaria antigen detection by bead-based assay and molecular confirmation by PCR shows no evidence of Pfhrp2 and Pfhrp3 deletion in Haiti

Abstract

Background: The Plasmodium falciparum parasite is the only human malaria that produces the histidine-rich protein 2 and 3 (HRP2/3) antigens. Currently, HRP2/3 are widely used in malaria rapid diagnostic tests (RDTs), but several global reports have recently emerged showing genetic deletion of one or both of these antigens in parasites. Deletion of these antigens could pose a major concern for P. falciparum diagnosis in Haiti which currently uses RDTs based solely on the detection of the HRP2/3 antigens.

Methods: From September 2012 through February 2014, dried blood spots (DBS) were collected in Haiti from 9317 febrile patients presenting to 17 health facilities in 5 departments throughout the country as part of a bed net intervention study. All DBS from RDT positive persons and a random sampling of DBS from RDT negative persons were assayed for P. falciparum DNA by nested and PET-PCR (n = 2695 total). All PCR positive samples (n = 331) and a subset of PCR negative samples (n = 95) were assayed for three malaria antigens by a multiplex bead assay: pan-Plasmodium aldolase (pAldo), pan-Plasmodium lactate dehydrogenase (pLDH), and HRP2/3. Any samples positive for P. falciparum DNA, but negative for HRP2/3 antigens were tested by nested PCR for Pfhrp2 and Pfhrp3 gene deletions.

Results: Of 2695 DBS tested for Plasmodium DNA, 345 (12.8%) were originally found to be positive for P. falciparum DNA; 331 of these had DBS available for antigen detection. Of these, 266 (80.4%) were positive for pAldo, 221 (66.8%) positive for pLDH, and 324 (97.9%) were positive for HRP2/3 antigens. Seven samples (2.1%) positive for P. falciparum DNA were not positive for any of the three antigens by the bead assay, and were investigated for potential Pfhrp2/3 gene deletion by PCR. These samples either successfully amplified Pfhrp2/3 genes or were at an estimated parasite density too low for sufficient DNA to perform successful genotyping.

Conclusions: Malaria positive samples in multiple Haitian sites were found to contain the HRP2/3 antigens, and no evidence was found of Pfhrp2/3 deletions. Malaria RDTs based on the detection of the HRP2/3 antigens remain a reliable P. falciparum diagnostic tool as Haiti works towards malaria elimination.

Keywords: HRP2 deletion; Haiti; Pfhrp2; Pfhrp3; Plasmodium aldolase; Plasmodium lactate dehydrogenase; Rapid diagnostic test.

Fig. 1

Departments of Haiti and locations of health facilities providing the 331 P. falciparum positive dried blood spots. Health facilities shown as dots on the map, and number of PET-PCR positive samples from each facility indicated by the number

Fig. 2

Different profiles of antigen positivity for 331 samples found to be PCR positive for P. falciparum DNA. Numbers indicate concordance of antigen positivity for all samples

Fig. 3

RDT and antigen positivity as a function of PCR-determined parasite density. Non-parametric (LOESS, blue lines) and parametric (logistic, red lines) regression of probability of single antigen positivity as a modeled by estimated parasite density. Outputs at selected probabilities displayed in Table 1

Fig. 4

Positivity of combinations of antigen positivity as a function of parasite density. Non-parametric (LOESS, blue lines) and parametric (logistic, red lines) regression of probability of antigen positivity as a modeled by estimated parasite density. Outputs at selected probabilities displayed in Table 1

Fig. 5

Sensitivity of HRP2-based RDT as a function of HRP2 concentration. Non-parametric (LOESS, blue line) and parametric (logistic, red line) regression of probability of RDT positivity as a modeled by HRP2 antigen concentration in person’s blood sample. Outputs at selected probabilities displayed in Table 1