International Journal for Parasitology 47 (2017) 379–383

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International Journal for Parasitology

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Invited Review

The unhealthy attraction of Plasmodium vivax to reticulocytes expressingtransferrin receptor 1 (CD71)

http://dx.doi.org/10.1016/j.ijpara.2017.03.0010020-7519/� 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail address: b.russell@otago.ac.nz (B. Russell).

Benoit Malleret a,b, Laurent Rénia a, Bruce Russell c,⇑a Singapore Immunology Network (SIgN), A*STAR, 8A Biomedical Grove, SingaporebDepartment of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 5 Science Drive 2, BlkMD4, Level 3, Singapore 117597, SingaporecDepartment of Microbiology and Immunology, University of Otago, Dunedin, New Zealand

a r t i c l e i n f o a b s t r a c t

Article history:Received 15 December 2016Received in revised form 28 March 2017Accepted 29 March 2017Available online 13 April 2017

Keywords:Plasmodium vivaxReticulocytesMerozoite invasionTransferrin receptor (CD71)Duffy Antigen Chemokine Receptor (CD234)

The majority of malaria parasite species prefer to invade reticulocytes, the most infamous beingPlasmodium vivax. While the absence of an in vitro continuous culture method has hampered the studyof P. vivax invasion biology, studies utilising primate models and ex vivo assays have provided someimportant insights. Most importantly, P. vivax merozoites have a strong preference for a subset of imma-ture erythrocytes characterised by the expression of the transferrin receptor (CD71). This current opinionpiece on P. vivax merozoite invasion highlights important gaps in our understanding of how this parasiterecognises and enters reticulocytes, and discusses some recent conceptual advances in P. vivax invasionbiology.

� 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

1. Reticulocytes are commonly targeted by malaria parasites

The intra-erythrocytic environment provides parasitic microor-ganisms with a nutrient rich and immunologically privileged envi-ronment for growth and reproduction. Consequently,haemoprotozoans such as Plasmodium spp., Babesia spp. and Hae-mogregarina spp. are some of the most successful and diverse par-asites, found in almost every phylogenetic class of vertebrate(Garnham, 1966). One of the disadvantages of targeting erythro-cytes is that they have a finite life span, thus inadvertently invad-ing senescent erythrocytes may result in premature clearance.Therefore, it is hardly surprising that most Plasmodium spp. preferto invade younger erythrocytes, allowing for uninterrupted asexualdevelopment and the extended circulation of sexual forms(Hegner, 1938; Chwatt, 1948; Landau and Killick-Kendrick, 1966;Coatney et al., 1971; Martin-Jaular et al., 2013). Certainly carefulinvestigation of primate malaria parasites reveals that most ofthem have a tropism for young and immature erythrocytes (retic-ulocytes) (Coatney et al., 1971). Even those Plasmodium spp. withthe capacity to invade a wide range of erythrocytes, such as Plas-modium falciparum, will preferentially invade reticulocytes if giventhe opportunity (Shushan et al., 1937; Wilson et al., 1977; Pasvol

et al., 1980). Of malaria parasite species with a strict tropism forreticulocytes, the most well-known is Plasmodium vivax (Craik,1920; Kitchen, 1938).

2. Reticulocytes are a heterogeneous population of erythrocytes

Before exploring how P. vivax merozoites invade reticulocytes,it is important to have a background understanding of reticulocytebiology. The term reticulocyte is a catch-all term for all immatureenucleated erythrocytes that still contain reticular matter (which isusually disclosed using supra/sub-vital stains such as new methy-lene blue or Giemsa (Lee et al., 2013). Far from being a homoge-nous set of red blood cells (RBCs), the approximately 90 h periodof reticulocyte development is characterised by a range of signifi-cant rheological, biochemical and immunological changes (Chasiset al., 1989; Chasis and Schrier, 1989). Early stage reticulocytesfound in the mammalian bone marrow are large, rigid, irregularspherical cells. As the reticulocytes mature they become smaller,biconcave and are subject to a qualitative and quantitative loss ofmembrane proteins (Malleret et al., 2013). The diverse nature ofreticulocytes was observed in the 1930s by Heilmeyer et al.(1932) and were grouped into four classes (Heilmeyer Class (HC)I, II, III and IV) (Heilmeyer andWesthauser, 1932). Early stage retic-ulocytes HC I and II are restricted to the bone marrow, HC III arefound mostly in the bone marrow and the most mature reticulo-

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cytes, HC IV, are the most common form of reticulocyte in theperipheral blood supply (Seip, 1953). Recent advances in flowcytometry have helped in the rapid quantification and classifica-tion of reticulocytes (Davis et al., 1995; Nobes and Carter, 1990).Perhaps the most important method utilises the transferrin recep-tor (CD71) which is sequentially lost as the reticulocyte matures(Kono et al., 2009; Malleret et al., 2013). While HC I to III stillexpress CD71 (CD71+), the HC IV reticulocytes have little or noCD71 (CD71�). The distinction between CD71+ and CD71� reticu-locytes is important as P. vivaxmerozoites have a strong preferencefor CD71+ reticulocytes. Importantly, the quantity of CD71 has lit-tle impact on invasion rates, only the presence or absence of CD71affects invasion rates.

3. Plasmodium vivax rapidly remodels the immunophenotype,intracellular content and nanostructure of the infectedreticulocyte

Before we discuss the detail of P. vivax invasion of CD71+ retic-ulocytes, it is important to deal with the previously reported asser-tion that P. vivax can invade mature RBCs (CD71� erythrocytes andnormocytes). In the early literature (Hegner, 1938) it was observedthat P. vivax parasites were found in mature RBCs (lacking anyreticular matter). The seemingly obvious conclusion from thesestudies is that P. vivax can also invade mature RBCs. However, care-ful longitudinal studies of P. vivax patient haematology revealedthat the loss of reticulate matter in most infected RBCs occurredapproximately 3 h post invasion (Vryonis, 1939). Some 70 yearslater, this observation was supported by controlled ex vivo studiesdemonstrating that in P. vivax-infected reticulocytes, reticular mat-ter was eliminated together with CD71 3 h post invasion (Malleretet al., 2015). Thus while they seem to be ‘infected normocytes’,they are simply reticulocytes subject to a rapid parasite-induceddestruction of reticular matter (Fig. 1). Perhaps the most interest-

Fig. 1. The invasion and development of Plasmodium vivax in transferrin receptor 1 (CD7in serial observations of ex vivo matured P. vivax (supravitally stained with Giemsa). Therroneous impression they have invaded normocytes.

ing change observed in the early stages of reticulocyte infectionare parasite driven changes to the RBC immunophenotype (lossof RBC receptors such as CD71). While we have only quantifiedCD71 loss post invasion (Malleret et al., 2015), we suspect that arange of other receptors are also lost, including those that are crit-ical for invasion (conceivably blocking the entry of other mero-zoites not simultaneously invading with the primary merozoite).How the reticular matter and transferrin receptors are removedis still a matter for conjecture; however the presence of numerousmicrovesicles appearing at the surface of the infected RBC (iRBC)soon after invasion may indicate that some host cell componentsare ejected (Malleret et al., 2015).

4. How does P. vivax specifically target immature reticulocytes?

While free P. vivax merozoites probably use P. vivax MerozoiteSurface Protein 1 (pvMSP1) to ‘sense’ the surface of erythrocytes(Chandramohanadas et al., 2014), how it specifically identifies areceptive immature reticulocyte is still a mystery (Fig. 2). The bestknown ligand receptor combination in P. vivax invasion biology isthe Duffy Binding Protein (PvDBP) – Duffy Antigen ChemokineReceptor (DARC or Duffy receptor) (Miller et al., 1976). AlthoughDuffy dependence is a key feature of P. vivax invasion (with someexceptions (Mendes et al., 2011; Wurtz et al., 2011; Woldearegaiet al., 2013)), the fact that DARC is also expressed on mature ery-throcytes (Malleret et al., 2013) means it is unlikely to be ‘the retic-ulocyte receptor’.

In the search for the P. vivax reticulocyte ‘sensor’, increasingattention has focussed on two families of merozoite proteinsknown as the reticulocyte binding proteins (RBPs) and erythrocytebinding proteins (EBPs) (Galinski et al., 1992; Rayner et al., 2001;Hester et al., 2013). While much has been done to characterisethe polymorphisms in these proteins, little is known about theircorresponding receptors or even if they are really reticulocyte-

1+) reticulocytes results in the rapid expulsion of reticular material (<3 h) as showne presence of tiny P. vivax rings in erythrocytes with no reticular matter gives the

Fig. 2. A representation of the process by which Plasmodium vivaxmerozoites invade transferrin receptor 1 (CD71+) reticulocytes. The early phases of erythrocyte recognitionare most likely mediated by P. vivax Merozoite Surface Protein 1 (pvMSP1). It is postulated that one of the Reticulocyte Binding Proteins (pvRBP) and Erythrocyte BindingProteins (EBPs) are then involved in the recognition of the reticulocyte (these receptors are not known(Cluster of Differentiation(CD?))). Finally, the Duffy Binding Protein(pvDBP)–Duffy Antigen Receptor for Chemokines (DARC) is most likely involved in end stages of merozoite invasion. Colours are used to indicate matching host receptor- P.vivax ligand combinations. ICAM-4, InterCellular Adhesion Molecule-4; GPA, Glycophorin A.

Fig. 3. Factors affecting the ex vivo invasion of reticulocytes by Plasmodium vivax merozoites (A) Inhibition of P. vivax invasion after chymotrypsin, neurominidase or trypsintreatment of the reticulocytes. Only chymotrypsin treatment inhibits P. vivax merozoite invasion. (B) Comparison of P. vivax invasion in reticulocytes from cord blood versusadult peripheral blood. Please refer to Supplementary Data S1 for the associated methodology.

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specific (Gruszczyk et al., 2016). To search for the correspondingRBP receptors is challenging, however below we outline some datathat may help in short listing candidate reticulocyte-specificreceptors.

5. The primary P. vivax invasion receptor is trypsin-resistantand restricted to CD71+ reticulocytes

Perhaps the most straightforward approach to discover the pri-mary receptor for P. vivax invasion is to compare the proteomes of

CD71+ and CD71� reticulocytes. A short list of receptors specific toCD71+ reticulocytes with extracellular domains provides a solidbasis for future discoveries. As P. vivax invasion is not affected bytreating reticulocytes with trypsin or neuraminidase (however,invasion is chymotrypsin-sensitive) (Barnwell et al., 1989)(Fig. 3A), the short list could be further narrowed down to thosereceptors resistant to trypsin. Please note that when consideringthe enzymatic profile of candidate receptors, it is important to alsoremember some receptors associated with Clathrin pits (Aikawaet al., 1975) may be continually recycled, thus confounding

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attempts at enzymatic treatment of those receptors (trypsin-sensitive CD71 is an example (Turkewitz et al., 1988)) (Fig. 1). Oncea suitably small number of putative receptors is identified, theycan be blocked by corresponding antibodies/recombinant proteinsand tested in a P. vivax ex vivo invasion inhibition assay. Such anti-body blocking studies may be confounded if the target receptor isassociated with a cluster of receptors such as the R4.1 receptorcomplex (Salomao et al., 2008). This is particularly important asDARC is located in these clusters (Fig. 2), and attempts to blockthe targeted receptor may result in the unintended steric hin-drance of the DARC receptor. If possible the expression of the puta-tive receptor should be knocked down using reticulocytesgenerated from transfected erythroid progenitors.

6. A note on cord blood versus adult reticulocytes

Any investigation into P. vivax merozoite invasion will use sig-nificant quantities of reticulocytes. The majority of our studieshave utilized cord blood as it is easy to obtain and is rich inCD71+ reticulocytes. Of course one perennial concern regards theuse of foetal versus adult reticulocytes. While we have not con-ducted a full proteomic comparison of adult and cord blood retic-ulocytes, our invasion studies indicate that there is no significantdifference in the invasion profile between these two groups(Fig. 3B). Neither is there any discernible effect of blood group (A,B or O) or the presence of foetal haemoglobin on P. vivax invasion(Russell et al., 2011). The major difference between adult and cordblood is the proportion of CD71+ versus CD71� reticulocytes. Thusattempts to purify CD71+ reticulocytes from non-anaemic volun-teers require large blood draws for reticulocyte selection. Wetherefore recommend the use of cord blood or in vitro culturedreticulocytes for serious investigations into P. vivax invasionbiology.

7. Future directions

One of the major impediments affecting future empirical stud-ies is the lack of a continuous culture system for P. vivax. WhileNew World primate models such as Aotus spp. (Porter, 1971) pro-vide a longer term solution for the provision of standard P. vivaxstrains, the cost and ethical pressures associated with these sys-tems is prohibitive. A mouse model producing human reticulocytesmay also offer some utility to study P. vivax; however these modelsare often as expensive as the primate models and produce a farlower yield of parasites than non-human primates (Kaushanskyet al., 2014). While human volunteer models are proving excitingand more accessible than previously thought (McCarthy et al.,2013), there are considerable ethical challenges associated withthe use of genetically modified reticulocytes and parasites inhumans. Perhaps our best chance to study P. vivax reticulocyteinvasion is to use its sister species, Plasmodium cynomolgi, whichis pathobiologically similar (Joyner et al., 2016) and can be culturedcontinuously (Nguyen-Dinh et al., 1981). The use of such a modelwould be particularly valuable if P. cynomolgi were humanised.

When the primary reticulocyte receptor is identified, it is hopedthat this receptor will be expressed or coated on normocytes tofacilitate the development of P. vivax continuous culture.

Acknowledgements

This study received funding from University of Otago, Dunedin,New Zealand Start-up Grant to Bruce Russell, Singapore NationalMedical Research Council (NMRC/CBRG/0047/2013) and from aYoung Investigator Grant (BMRC YIG Grant No:13/1/16/YA/009)to Benoit Malleret under the Agency for Science, Technology and

Research (A⁄STAR, Singapore). We thank Prof. Georges Snounou(Sorbonne Universités, UPMC Univ Paris 06, Inserm, France), foruseful discussions on P. vivax invasion.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ijpara.2017.03.001.

References

Aikawa, M., Miller, L.H., Rabbege, J., 1975. Caveola-vesicle complexes in theplasmalemma of erythrocytes infected by Plasmodium vivax and P. cynomolgi.Unique structures related to Schüffner’s dots. Am. J. Pathol. 79, 285–300.

Barnwell, J.W., Nichols, M.E., Rubinstein, P., 1989. In vitro evaluation of the role ofthe Duffy blood group in erythrocyte invasion by Plasmodium vivax. J. Exp. Med.169, 1795–1802.

Chandramohanadas, R., Russell, B., Liew, K., Yau, H.Y., Chong, A., Min, L., Gunalan, K.,Raman, R., Renia, L., Nosten, F., Shochat, S.G., Dao, M., Sasisekharan, R., Suresh,S., Preiser, P.R., 2014. Small molecule targeting Malaria merozoite surfaceprotein-1 (MSP-1) prevent host invasion of divergent plasmodial species. J.Infect. Dis. 210, 1616–1626.

Chasis, J.A., Prenant, M., Leung, A., Mohandas, N., 1989. Membrane assembly andremodeling during reticulocyte maturation. Blood 74, 1112–1120.

Chasis, J.A., Schrier, S.L., 1989. Membrane deformability and the capacity for shapechange in the erythrocyte. Blood 74, 2562–2568.

Chwatt, L.J., 1948. Infection of reticulocytes by Plasmodium falciparum andPlasmodium malariae in hyperendemic indigenous malaria. Ann. Trop. Med.Parasitol. 42, 101–112.

Coatney, G.R., Collins, W.E., Warren, M., Contacos, P.G., 1971. The Primate Malarias.Government Printing Office, Washington DC, U.S.A..

Craik, R., 1920. A note on the erythrocytes in malaria. Lancet 195, 1110.Davis, B.H., Ornvold, K., Bigelow, N.C., 1995. Flow cytometric reticulocyte maturity

index: a useful laboratory parameter of erythropoietic activity in anemia.Cytometry 22, 35–39.

Galinski, M.R., Medina, C.C., Ingravallo, P., Barnwell, J.W., 1992. A reticulocyte-binding protein complex of Plasmodium vivax merozoites. Cell 69, 1213–1226.

Garnham, P.C.C., 1966. Malaria Parasites and Other Haemosporidia. BlackwellScientific Publications, Oxford.

Gruszczyk, J., Lim, N.T., Arnott, A., He, W.Q., Nguitragool, W., Roobsoong, W., Mok, Y.F., Murphy, J.M., Smith, K.R., Lee, S., Bahlo, M., Mueller, I., Barry, A.E., Tham, W.H., 2016. Structurally conserved erythrocyte-binding domain in Plasmodiumprovides a versatile scaffold for alternate receptor engagement. Proc. Natl. Acad.Sci. U.S.A. 113, E191–E200.

Hegner, R., 1938. Relative frequency of ring-stage plasmodia in reticulocytes andmature erythrocytes in man an monkey. Am. J. Trop. Med. Hyg. 27, 690–718.

Heilmeyer, L., Westhauser, R., 1932. Reifungsstadien an ÜberlebendenReticulozyten In Vitro und ihre Bedeutung für die Schaetzung der täglichenHaemoglobin-Produktion In Vivo. Ztschr. klin. Med. 121, 361–379.

Hester, J., Chan, E.R., Menard, D., Mercereau-Puijalon, O., Barnwell, J., Zimmerman, P.A., Serre, D., 2013. De novo assembly of a field isolate genome reveals novelPlasmodium vivax erythrocyte invasion genes. PLoS Neglect. Trop. Dis. 7, e2569.

Joyner, C., Moreno, A., Meyer, E.V., Cabrera-Mora, M., Kissinger, J.C., Barnwell, J.W.,Galinski, M.R., 2016. Plasmodium cynomolgi infections in rhesus macaquesdisplay clinical and parasitological features pertinent to modelling vivaxmalaria pathology and relapse infections. Malar. J. 15, 451.

Kaushansky, A., Mikolajczak, S.A., Vignali, M., Kappe, S.H., 2014. Of men in mice: thesuccess and promise of humanized mouse models for human malaria parasiteinfections. Cell Microbiol. 16, 602–611.

Kitchen, S.F., 1938. The infection of reticulocytes by Plasmodium vivax. Am. J. Trop.Med. 18, 347–359.

Kono, M., Kondo, T., Takagi, Y., Wada, A., Fujimoto, K., 2009. Morphologicaldefinition of CD71 positive reticulocytes by various staining techniques andelectron microscopy compared to reticulocytes detected by an automatedhematology analyzer. Clin. Chim. Acta 404, 105–110.

Landau, I., Killick-Kendrick, R., 1966. Rodent plasmodia of the RepubliqueCentrafricaine: the sporogony and tissue stages of Plasmodium chabaudi and P.berghei yoelii. Trans. R. Soc. Trop. Med. Hyg. 60, 633–649.

Lee, W.C., Russell, B., Lau, Y.L., Fong, M.Y., Chu, C., Sriprawat, K., Suwanarusk, R.,Nosten, F., Renia, L., 2013. Giemsa-stained wet mount based method forreticulocyte quantification: a viable alternative in resource limited or malariaendemic settings. PLoS One 8, e60303.

Malleret, B., Li, A., Zhang, R., Tan, K.S., Suwanarusk, R., Claser, C., Cho, J.S., Koh, E.G.,Chu, C.S., Pukrittayakamee, S., Ng, M.L., Ginhoux, F., Ng, L.G., Lim, C.T., Nosten, F.,Snounou, G., Renia, L., Russell, B., 2015. Plasmodium vivax: restricted tropismand rapid remodeling of CD71-positive reticulocytes. Blood 125, 1314–1324.

Malleret, B., Xu, F., Mohandas, N., Suwanarusk, R., Chu, C., Leite, J.A., Low, K., Turner,C., Sriprawat, K., Zhang, R., Bertrand, O., Colin, Y., Costa, F.T., Ong, C.N., Ng, M.L.,Lim, C.T., Nosten, F., Renia, L., Russell, B., 2013. Significant biochemical,biophysical and metabolic diversity in circulating human cord bloodreticulocytes. PLoS One 8, e76062.

B. Malleret et al. / International Journal for Parasitology 47 (2017) 379–383 383

Martin-Jaular, L., Elizalde-Torrent, A., Thomson-Luque, R., Ferrer, M., Segovia, J.C.,Herreros-Aviles, E., Fernandez-Becerra, C., Del Portillo, H.A., 2013. Reticulocyte-prone malaria parasites predominantly invade CD71hi immature cells:implications for the development of an in vitro culture for Plasmodium vivax.Malar. J. 12, 434.

McCarthy, J.S., Griffin, P.M., Sekuloski, S., Bright, A.T., Rockett, R., Looke, D., Elliott, S.,Whiley, D., Sloots, T., Winzeler, E.A., Trenholme, K.R., 2013. Experimentallyinduced blood-stage Plasmodium vivax infection in healthy volunteers. J. Infect.Dis. 208, 1688–1694.

Mendes, C., Dias, F., Figueiredo, J., Mora, V.G., Cano, J., de Sousa, B., do Rosario, V.E.,Benito, A., Berzosa, P., Arez, A.P., 2011. Duffy negative antigen is no longer abarrier to Plasmodium vivax - molecular evidence from the African West Coast(Angola and Equatorial Guinea). PLoS Negl. Trop. Dis. 5, e1192.

Miller, L.H., Mason, S.J., Clyde, D.F., McGinniss, M.H., 1976. The resistance factor toPlasmodium vivax in blacks. The Duffy-blood-group genotype, FyFy. N. Engl. J.Med. 295, 302–304.

Nguyen-Dinh, P., Gardner, A.L., Campbell, C.C., Skinner, J.C., Collins, W.E., 1981.Cultivation in vitro of the vivax-type malaria parasite Plasmodium cynomolgi.Science 212, 1146–1148.

Nobes, P.R., Carter, A.B., 1990. Reticulocyte counting using flow cytometry. J. Clin.Pathol. 43, 675–678.

Pasvol, G., Weatherall, D.J., Wilson, R.J., 1980. The increased susceptibility of youngred cells to invasion by the malarial parasite Plasmodium falciparum. Br. J.Haematol. 45, 285–295.

Porter Jr., J.A., 1971. Plasmodium vivax infections in Aotus trivirgatus. Am. J. Trop.Med. Hyg. 20, 535–538.

Rayner, J.C., Vargas-Serrato, E., Huber, C.S., Galinski, M.R., Barnwell, J.W., 2001. APlasmodium falciparum homologue of Plasmodium vivax reticulocyte bindingprotein (PvRBP1) defines a trypsin-resistant erythrocyte invasion pathway. J.Exp. Med. 194, 1571–1581.

Russell, B., Suwanarusk, R., Borlon, C., Costa, F.T., Chu, C.S., Rijken, M.J., Sriprawat, K.,Warter, L., Koh, E.G., Malleret, B., Colin, Y., Bertrand, O., Adams, J.H.,D’Alessandro, U., Snounou, G., Nosten, F., Renia, L., 2011. A reliable ex vivoinvasion assay of human reticulocytes by Plasmodium vivax. Blood 118, e74–e81.

Salomao, M., Zhang, X., Yang, Y., Lee, S., Hartwig, J.H., Chasis, J.A., Mohandas, N., An,X., 2008. Protein 4.1R-dependent multiprotein complex: new insights into thestructural organization of the red blood cell membrane. Proc. Natl. Acad. Sci. U.S.A. 105, 8026–8031.

Seip, M., 1953. Chapter V: The reticulocyte maturation in the peripheral blood. ActaMed. Scand. 146, 49–69.

Shushan, M., Blitz, O., Adams, C.G., 1937. The role of reticulocytes in malaria. J. Lab.Clin. Med. 22, 364–370.

Turkewitz, A.P., Amatruda, J.F., Borhani, D., Harrison, S.C., Schwartz, A.L., 1988. Ahigh yield purification of the human transferrin receptor and properties of itsmajor extracellular fragment. J. Biol. Chem. 263, 8318–8325.

Vryonis, G., 1939. Observations on the parasitization of erythrocytes by Plasmodiumvivax, with special reference to reticulocytes. Am. J. Trop. Med. Hyg. 30, 41–48.

Wilson, R.J., Pasvol, G., Weatherall, D.J., 1977. Invasion and growth of Plasmodiumfalciparum in different types of human erythrocyte. Bull. World Health Organ.55, 179–186.

Woldearegai, T.G., Kremsner, P.G., Kun, J.F., Mordmuller, B., 2013. Plasmodium vivaxmalaria in Duffy-negative individuals from Ethiopia. Trans. R. Soc. Trop. Med.Hyg. 107, 328–331.

Wurtz, N., Mint Lekweiry, K., Bogreau, H., Pradines, B., Rogier, C., Ould MohamedSalem Boukhary, A., Hafid, J.E., Ould Ahmedou Salem, M.S., Trape, J.F., Basco, L.K.,Briolant, S., 2011. Vivax malaria in Mauritania includes infection of a Duffy-negative individual. Malar. J. 10, 336.