histidine-rich protein 2 (PfHRP2) forms the basis of many current malaria rapid diagnostic tests (RDTs). However, the parasites lacking part or all of the gene do not express the PfHRP2 protein and are, therefore, not identifiable by PfHRP2-detecting RDTs. We evaluated the performance of the SD Bioline Malaria Ag P.f/Pan RDT together with variation in Madagascar. Genomic DNA isolated from 260 patient blood samples were polymerase chain reaction (PCR)-amplified for the parasite 18S rRNA and genes. Post-PCR ligation detection reaction-fluorescent microsphere assay (LDR-FMA) was performed for the identification of parasite species. histidine-rich protein 2 amplicons were sequenced. Polymerase chain reaction diagnosis of patient samples showed that 29% (75/260) were infected and was present in 95% (71/75) of these PCR-positive samples. Comparing RDT and detection by LDR-FMA, eight samples were RDT negative but positive (false negatives), all of which were positive. The sensitivity and specificity of the RDT were 87% and 90%, respectively. Seventy-three samples were amplified for , from which nine randomly selected amplicons were sequenced, yielding 13 sequences. Amplification of, combined with RDT analysis and detection by LDR-FMA, showed that there was no indication of deletion. Sequence analysis of showed that the correlation between sequence structure and RDT detection rates was unclear. Although the observed absence of deletion from the samples screened here is encouraging, continued monitoring of the efficacy of the SD Bioline Malaria Ag P.f/Pan RDT for malaria diagnosis in Madagascar is warranted.

Histidine-rich protein 2 of (PfHRP2) forms the basis of many current malaria rapid diagnostic tests (RDTs). It is concerning that there are parasites that lack part or all of the gene, and thus do not express the PfHRP2 protein; such parasites are not identifiable by PfHRP2-detecting RDTs. Very limited information is available regarding genetic variation in Papua New Guinea (PNG). In the present study, this gene variation was evaluated using 169 samples previously collected from the Wosera area in East Sepik Province of PNG. Molecular diagnosis of these samples showed that 81% were infected, and was present in 91% of those infected samples. One hundred and twenty samples were amplified for exon-2, from which 12 randomly selected amplicons were sequenced, yielding 18 sequences, all of which were unique. Baker repeat type 2 × type 7 numbers ranged from 0 to 108. Epitope mapping analysis revealed that three major epitopes, DAHHAHHA, AHHAADAHHA, and AHHAADAHH, were present in high prevalence and frequencies. These major epitopes have been shown to be recognized by the monoclonal antibodies 3A4 and PTL-3 (DAHHAHHA), C1-13 (AHHAADAHHA), and S2-5 and C2-3 (AHHAADAHH). This study provides further information on the high genetic variation of and its unclear relationship with prediction of RDT detection sensitivity, and identifies major epitopes in this gene from PNG. These results could be relevant and useful to understand the genetic diversity of this gene and the performance of current and future RDTs in this malarious region of the world.