Rising costs of antimalarial agents are increasing the demand for accurate diagnosis of malaria. Rapid diagnostic tests (RDTs) offer great potential to improve the diagnosis of malaria, particularly in remote areas. Many RDTs are based on the detection of Plasmodium falciparum histidine-rich protein (PfHRP) 2, but reports from field tests have questioned their sensitivity and reliability. We hypothesize that the variability in the results of PfHRP2-based RDTs is related to the variability in the target antigen. We tested this hypothesis by examining the genetic diversity of PfHRP2, which includes numerous amino acid repeats, in 75 P. falciparum lines and isolates originating from 19 countries and testing a subset of parasites by use of 2 PfHRP2-based RDTs. We observed extensive diversity in PfHRP2 sequences, both within and between countries. Logistic regression analysis indicated that 2 types of repeats were predictive of RDT detection sensitivity (87.5% accuracy), with predictions suggesting that only 84% of P. falciparum parasites in the Asia-Pacific region are likely to be detected at densities < or = 250 parasites/microL. Our data also indicated that PfHRP3 may play a role in the performance of PfHRP2-based RDTs. These findings provide an alternative explanation for the variable sensitivity in field tests of malaria RDTs that is not due to the quality of the RDTs.

BackgroundAccurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region.MethodsThe pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs.ResultsSequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified.ConclusionsThe results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation.

The ability to accurately diagnose malaria infections, particularly in settings where laboratory facilities are not well developed, is of key importance in the control of this disease. Rapid diagnostic tests (RDTs) offer great potential to address this need. Reports of significant variation in the field performance of RDTs based on the detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) (PfHRP2) and of significant sequence polymorphism in PfHRP2 led us to evaluate the binding of four HRP2-specific monoclonal antibodies (MABs) to parasite proteins from geographically distinct P. falciparum isolates, define the epitopes recognized by these MABs, and relate the copy number of the epitopes to MAB reactivity. We observed a significant difference in the reactivity of the same MAB to different isolates and between different MABs tested with single isolates. When the target epitopes of three of the MABs were determined and mapped onto the peptide sequences of the field isolates, significant variability in the frequency of these epitopes was observed. These findings support the role of sequence variation as an explanation for variations in the performance of HRP2-based RDTs and point toward possible approaches to improve their diagnostic sensitivities.

Artemisinin-derivative combination therapies (ACT) are highly efficacious against multidrug-resistant Plasmodium falciparum malaria. Few efficacy data, however, are available for vivax malaria. With high rates of chloroquine (CQ) resistance in both vivax and falciparum malaria in Papua Province, Indonesia, new combination therapies are required for both species. We recently found artesunate plus sulfadoxine-pyrimethamine (ART-SP) to be highly effective (96%) in the treatment of falciparum malaria in Papua Province. Following a preliminary study of CQ plus sulfadoxine-pyrimethamine (CQ-SP) for the treatment of Plasmodium vivax infection, we used modified World Health Organization criteria to evaluate the efficacy of ART-SP for the treatment of vivax malaria in Papua. Nineteen of 22 patients treated with ART-SP could be evaluated on day 28, with no early treatment failures. Adequate clinical and parasitological responses were found by day 14 in all 20 (100%) of the patients able to be evaluated and by day 28 in 17 patients (89.5%). Fever and parasite clearance times were short, with hematological improvement observed in 70.6% of the patients. Double (at positions 58 and 117) and quadruple (at positions 57, 58, 61, and 117) mutations in the P. vivax dihydrofolate reductase (PvDHFR) were common in Papuan P. vivax isolates (46 and 18%, respectively). Treatment failure with SP-containing regimens was significantly higher with isolates with this PvDHFR quadruple mutation, which included a novel T3M mutation at residue 61 linked to an S3T (but not an S3N) mutation at residue 117. ART-SP ACT resulted in a high cure rate for both major Plasmodium species in Papua, though progression of DHFR mutations in both species due to the continued use of SP monotherapy for clinically diagnosed malaria threatens the future utility of this combination.High rates of chloroquine (CQ) resistance and CQ treatment failure have been found in both vivax and falciparum malaria in Papua Province (formerly Irian Jaya), Indonesia (2,3,5,21,28,38), and new combination therapies are required for each species. Artemisinin-derivative combination therapies (ACT) are being used increasingly for the treatment of multidrug-resistant Plasmodium falciparum malaria (29) because of their excellent efficacy (23,31,39,41), their ability to slow or reverse the emergence of resistance (24, 30), and their ability to reduce malaria transmission (24, 30). Despite the coexistence of falciparum and vivax malaria in many parts of the world where malaria treatment is usually based on clinical diagnosis, there have been few studies assessing the efficacy of artemisinin derivatives in treating vivax malaria and none in the area of CQ-resistant P. vivax. Studies of artesunate (ART) monotherapy for P. vivax infection have demonstrated rapid clearance of fever and parasites (1,6,33,43). However, with its very short half-life, no ART remains by the time of the first relapse of P. vivax infection, approximately 3 weeks after treatment begins, resulting in a high frequency of...

Mutations in the pfcrt and pfmdr1 genes have been associated with chloroquine resistance in Plasmodium falciparum. Ten and five mutations, respectively, have been identified in these genes from chloroquine-resistant parasites worldwide. Mutation patterns in pfcrt revealed that chloroquine resistance evolved independently in southeast Asia, South America, and Papua New Guinea. However, the evolution of chloroquine resistance in the rest of the Pacific region is unclear. In this study, we examined sequence polymorphisms in these genes in isolates from Morong, Philippines, and compared them to known chloroquine resistance sequences. Two novel mutations, A144T and L160Y, were identified outside of the 10 known mutations in pfcrt in Morong isolates. These novel mutations were identified only in parasites with K76T and N326D but without the common A220S mutation found in most chloroquine-resistant isolates. This represents a unique chloroquine resistance allelic type (K76T/A144T/L160Y/N326D) not previously found elsewhere in the world. One Morong isolate also had an additional C72S mutation, whereas only one isolate possessed an allelic type typical of chloroquine resistance in Asia. Parasites with the novel pfcrt allelic types were resistant to chloroquine in vitro and were unresponsive to verapamil (0.9 M) chemosensitization, similar to chloroquine-resistant parasites from South America and Papua New Guinea. These results suggest that chloroquine resistance evolved independently in the Philippines and represents a second chloroquine resistance founder event in the South Pacific.