Volume 98, Issue 6
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645



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.


Article metrics loading...

The graphs shown below represent data from March 2017
Loading full text...

Full text loading...



  1. Parra ME, Evans CB, Taylor DW, , 1991. Identification of Plasmodium falciparum histidine-rich protein 2 in the plasma of humans with malaria. J Clin Microbiol 29: 16291634. [Google Scholar]
  2. Beadle C, Long GW, Weiss WR, McElroy PD, Maret SM, Oloo AJ, Hoffman SL, , 1994. Diagnosis of malaria by detection of Plasmodium falciparum HRP-2 antigen with a rapid dipstick antigen-capture assay. Lancet 343: 564568. [Google Scholar]
  3. Moody A, , 2002. Rapid diagnostic tests for malaria parasites. Clin Microbiol Rev 15: 6678. [Google Scholar]
  4. Rabarijaona LP, Ariey F, Matra R, Cot S, Raharimalala AL, Ranaivo LH, Le Bras J, Robert V, Randrianarivelojosia M, , 2006. Low autochtonous urban malaria in Antananarivo (Madagascar). Malar J 5: 27. [Google Scholar]
  5. Randrianasolo L, Tafangy PB, Raharimalala LA, Ratsimbasoa AC, Randriamanantena A, Randrianarivelojosia M, , 2007. Rapid diagnostic test for malaria: preliminary study in Madagascar in 2003. Sante 17: 6973. [Google Scholar]
  6. Howes RE, Mioramalala SA, Ramiranirina B, Franchard T, Rakotorahalahy AJ, Bisanzio D, Gething PW, Zimmerman PA, Ratsimbasoa A, , 2016. Contemporary epidemiological overview of malaria in Madagascar: operational utility of reported routine case data for malaria control planning. Malar J 15: 502. [Google Scholar]
  7. Rakotonirina H, Barnadas C, Raherijafy R, Andrianantenaina H, Ratsimbasoa A, Randrianasolo L, Jahevitra M, Andriantsoanirina V, Menard D, , 2008. Accuracy and reliability of malaria diagnostic techniques for guiding febrile outpatient treatment in malaria-endemic countries. Am J Trop Med Hyg 78: 217221. [Google Scholar]
  8. Ratsimbasoa A, Fanazava L, Radrianjafy R, Ramilijaona J, Rafanomezantsoa H, Menard D, , 2008. Evaluation of two new immunochromatographic assays for diagnosis of malaria. Am J Trop Med Hyg 79: 670672. [Google Scholar]
  9. Ravaoarisoa E, Andriamiandranoro T, Raherinjafy R, Jahevitra M, Razanatsiorimalala S, Andrianaranjaka V, Randrianarivelojosia M, , 2017. Evaluation of the OnSite malaria rapid test performance in Miandrivazo, Madagascar. Bull Soc Pathol Exot 110: 254259. [Google Scholar]
  10. Baker J, Gatton ML, Peters J, Ho MF, McCarthy JS, Cheng Q, , 2011. Transcription and expression of Plasmodium falciparum histidine-rich proteins in different stages and strains: implications for rapid diagnostic tests. PLoS One 6: e22593. [Google Scholar]
  11. Ho MF, 2014. Circulating antibodies against Plasmodium falciparum histidine-rich proteins 2 interfere with antigen detection by rapid diagnostic tests. Malar J 13: 480. [Google Scholar]
  12. Iqbal J, Siddique A, Jameel M, Hira PR, , 2004. Persistent histidine-rich protein 2, parasite lactate dehydrogenase, and panmalarial antigen reactivity after clearance of Plasmodium falciparum monoinfection. J Clin Microbiol 42: 42374241. [Google Scholar]
  13. Cheng Q, Gatton ML, Barnwell J, Chiodini P, McCarthy J, Bell D, Cunningham J, , 2014. Plasmodium falciparum parasites lacking histidine-rich protein 2 and 3: a review and recommendations for accurate reporting. Malar J 13: 283. [Google Scholar]
  14. Lee N, Baker J, Andrews KT, Gatton ML, Bell D, Cheng Q, McCarthy J, , 2006. Effect of sequence variation in Plasmodium falciparum histidine-rich protein 2 on binding of specific monoclonal antibodies: implications for rapid diagnostic tests for malaria. J Clin Microbiol 44: 27732778. [Google Scholar]
  15. Lee N, Gatton ML, Pelecanos A, Bubb M, Gonzalez I, Bell D, Cheng Q, McCarthy JS, , 2012. Identification of optimal epitopes for Plasmodium falciparum rapid diagnostic tests that target histidine-rich proteins 2 and 3. J Clin Microbiol 50: 13971405. [Google Scholar]
  16. Gamboa D, 2010. A large proportion of P. falciparum isolates in the Amazon region of Peru lack pfhrp2 and pfhrp3: implications for malaria rapid diagnostic tests. PLoS One 5: e8091. [Google Scholar]
  17. Akinyi Okoth S, 2015. Variation in Plasmodium falciparum histidine-rich protein 2 (pfhrp2) and Plasmodium falciparum histidine-rich protein 3 (pfhrp3) gene deletions in Guyana and Suriname. PLoS One 10: e0126805. [Google Scholar]
  18. Dorado EJ, Okoth SA, Montenegro LM, Diaz G, Barnwell JW, Udhayakumar V, Murillo Solano C, , 2016. Genetic characterisation of Plasmodium falciparum isolates with deletion of the pfhrp2 and/or pfhrp3 genes in Colombia: the Amazon region, a challenge for malaria diagnosis and control. PLoS One 11: e0163137. [Google Scholar]
  19. Murillo Solano C, 2015. Deletion of Plasmodium falciparum histidine-rich protein 2 (pfhrp2) and histidine-rich protein 3 (pfhrp3) genes in Colombian parasites. PLoS One 10: e0131576. [Google Scholar]
  20. Rachid Viana GM, 2017. Histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia. PLoS One 12: e0171150. [Google Scholar]
  21. Abdallah JF, 2015. Prevalence of pfhrp2 and pfhrp3 gene deletions in Puerto Lempira, Honduras. Malar J 14: 19. [Google Scholar]
  22. Bharti PK, Chandel HS, Ahmad A, Krishna S, Udhayakumar V, Singh N, , 2016. Prevalence of pfhrp2 and/or pfhrp3 gene deletion in Plasmodium falciparum population in eight highly endemic states in India. PLoS One 11: e0157949. [Google Scholar]
  23. Biswas S, Tomar D, Rao DN, , 2005. Investigation of the kinetics of histidine-rich protein 2 and of the antibody responses to this antigen, in a group of malaria patients from India. Ann Trop Med Parasitol 99: 553562. [Google Scholar]
  24. Nima MK, Hougard T, Hossain ME, Kibria MG, Mohon AN, Johora FT, Rahman R, Haque R, Alam MS, , 2017. Case report: a case of Plasmodium falciparum hrp2 and hrp3 gene mutation in Bangladesh. Am J Trop Med Hyg 97: 11551158. [Google Scholar]
  25. Atroosh WM, 2015. Genetic variation of pfhrp2 in Plasmodium falciparum isolates from Yemen and the performance of HRP2-based malaria rapid diagnostic test. Parasit Vectors 8: 388. [Google Scholar]
  26. Amoah LE, Abankwa J, Oppong A, , 2016. Plasmodium falciparum histidine rich protein-2 diversity and the implications for PfHRP 2: based malaria rapid diagnostic tests in Ghana. Malar J 15: 101. [Google Scholar]
  27. Koita OA, 2012. False-negative rapid diagnostic tests for malaria and deletion of the histidine-rich repeat region of the hrp2 gene. Am J Trop Med Hyg 86: 194198. [Google Scholar]
  28. Kozycki CT, Umulisa N, Rulisa S, Mwikarago EI, Musabyimana JP, Habimana JP, Karema C, Krogstad DJ, , 2017. False-negative malaria rapid diagnostic tests in Rwanda: impact of Plasmodium falciparum isolates lacking hrp2 and declining malaria transmission. Malar J 16: 123. [Google Scholar]
  29. Parr JB, 2017. Pfhrp2-deleted Plasmodium falciparum parasites in the Democratic Republic of the Congo: a national cross-sectional survey. J Infect Dis 216: 3644. [Google Scholar]
  30. Watson OJ, Slater HC, Verity R, Parr JB, Mwandagalirwa MK, Tshefu A, Meshnick SR, Ghani AC, , 2017. Modelling the drivers of the spread of Plasmodium falciparum hrp2 gene deletions in sub-Saharan Africa. Elife 6: e25008. [Google Scholar]
  31. Wurtz N, 2013. Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests. Malar J 12: 34. [Google Scholar]
  32. Baker J, 2010. Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests. Malar J 9: 129. [Google Scholar]
  33. Baker J, McCarthy J, Gatton M, Kyle DE, Belizario V, Luchavez J, Bell D, Cheng Q, , 2005. Genetic diversity of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and its effect on the performance of PfHRP2-based rapid diagnostic tests. J Infect Dis 192: 870877. [Google Scholar]
  34. Mariette N, Barnadas C, Bouchier C, Tichit M, Menard D, , 2008. Country-wide assessment of the genetic polymorphism in Plasmodium falciparum and Plasmodium vivax antigens detected with rapid diagnostic tests for malaria. Malar J 7: 219. [Google Scholar]
  35. Howes RE, Chan ER, Rakotomanga TA, Schulte S, Gibson J, Zikursh M, Franchard T, Ramiranirina B, Ratsimbasoa A, Zimmerman PA, , 2017. Prevalence and genetic variants of G6PD deficiency among two Malagasy populations living in Plasmodium vivax-endemic areas. Malar J 16: 139. [Google Scholar]
  36. Menard D, 2010. Plasmodium vivax clinical malaria is commonly observed in Duffy-negative Malagasy people. Proc Natl Acad Sci USA 107: 59675971. [Google Scholar]
  37. National Malaria Control Programme of Madagascar, 2015. Plan stratégique de lutte contre le paludisme Madagascar 2013–2017. Consolider les acquis en vue de l'élimination du paludisme à Madagascar. Version revisée pour 2015–2017.
  38. Normark J, , 2008. Freezing of patient isolates and strains with glycerolyte. Moll K, Ljungström I, Perlmann H, Scherf A, Wahlgren M, eds. Methods in Malaria Research. Manassas, VA: Malaria Research and Reference Reagent Resource Center, American Type Culture Collection, 14.
  39. Grimberg BT, Erickson JJ, Sramkoski RM, Jacobberger JW, Zimmerman PA, , 2008. Monitoring Plasmodium falciparum growth and development by UV flow cytometry using an optimized Hoechst-thiazole orange staining strategy. Cytometry A 73: 546554. [Google Scholar]
  40. Blomqvist K, , 2008. Thawing of glycerolyte-frozen parasites with NaCl. Moll K, Ljungström I, Perlmann H, Scherf A, Wahlgren M, eds. Methods in Malaria Research. Manassas, VA: Malaria Research and Reference Reagent Resource Center, American Type Culture Collection, 15.
  41. McNamara DT, Kasehagen LJ, Grimberg BT, Cole-Tobian J, Collins WE, Zimmerman PA, , 2006. Diagnosing infection levels of four human malaria parasite species by a polymerase chain reaction/ligase detection reaction fluorescent microsphere-based assay. Am J Trop Med Hyg 74: 413421. [Google Scholar]
  42. Walker-Jonah A, Dolan SA, Gwadz RW, Panton LJ, Wellems TE, , 1992. An RFLP map of the Plasmodium falciparum genome, recombination rates and favored linkage groups in a genetic cross. Mol Biochem Parasitol 51: 313320. [Google Scholar]
  43. Sullivan DJ, Jr Ayala YM, Goldberg DE, , 1996. An unexpected 5′ untranslated intron in the P. falciparum genes for histidine-rich proteins II and III. Mol Biochem Parasitol 83: 247251. [Google Scholar]
  44. Zimmerman PA, Howes RE, , 2015. Malaria diagnosis for malaria elimination. Curr Opin Infect Dis 28: 446454. [Google Scholar]
  45. Rice BL, Golden CD, Anjaranirina EJ, Botelho CM, Volkman SK, Hartl DL, , 2016. Genetic evidence that the Makira region in northeastern Madagascar is a hotspot of malaria transmission. Malar J 15: 596. [Google Scholar]
  46. Trouvay M, Palazon G, Berger F, Volney B, Blanchet D, Faway E, Donato D, Legrand E, Carme B, Musset L, , 2013. High performance of histidine-rich protein 2 based rapid diagnostic tests in French Guiana are explained by the absence of pfhrp2 gene deletion in P. falciparum. PLoS One 8: e74269. [Google Scholar]
  47. Sharma YD, , 1988. Genomic organization, structure and possible function of histidine-rich proteins of malaria parasites. Int J Biochem 20: 471477. [Google Scholar]
  48. Mackay M, Goman M, Bone N, Hyde JE, Scaife J, Certa U, Stunnenberg H, Bujard H, , 1985. Polymorphism of the precursor for the major surface antigens of Plasmodium falciparum merozoites: studies at the genetic level. EMBO J 4: 38233829. [Google Scholar]
  49. Ramutton T, 2012. Sequence variation does not confound the measurement of plasma PfHRP2 concentration in African children presenting with severe malaria. Malar J 11: 276. [Google Scholar]
  50. Leow CH, Jones M, Cheng Q, Mahler S, McCarthy J, , 2014. Production and characterization of specific monoclonal antibodies binding the Plasmodium falciparum diagnostic biomarker, histidine-rich protein 2. Malar J 13: 277. [Google Scholar]
  51. Ravaoarisoa E, Zamanka H, Fusai T, Bellalou J, Bedouelle H, Mercereau-Puijalon O, Fandeur T, , 2010. Recombinant antibodies specific for the Plasmodium falciparum histidine-rich protein 2. MAbs 2: 416427. [Google Scholar]

Data & Media loading...

Supplemental Table

  • Received : 31 Oct 2017
  • Accepted : 20 Dec 2017
  • Published online : 19 Mar 2018

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error