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AcknowledgementsThe atlas is a ‘work in progress’ and hence the estimates provided arepreliminary. Revision and improvement require further information onthe prevalence of infection within countries. If you know of relevant datathat could be included, or if you would like to be a partner in this initia-tive, then please contact the WHO in Geneva. Collaborating partnersinclude the Strategy Development and Monitoring for Parasitic Diseasesand Vector Control, Communicable Disease Control, Prevention andEradication, World Health Organization, 1211, Geneva 27 (Dirk Engels,Lester Chitsulo and Antonio Montreso); the WHO/UNICEF JointProgramme on Health Mapping and GIS, HealthMap (Kathy O’Neill,Jean-Pierre Meert and Isabelle Nutall); and the Partnership for ChildDevelopment and the International School Health Initiative of the WorldBank. The Partnership is supported by the United Nations DevelopmentProgramme, the Rockefeller Foundation, the Edna McConnell ClarkFoundation, the James S. McDonnell Foundation, the Wellcome Trust,the World Bank, UNICEF, and the WHO. SB is in receipt of a WellcomeTrust Prize Studentship. DAPB acknowledges the financial support of theWellcome Trust. We are grateful to Andrew Hall, Simon Hay and EdwinMichael for useful discussions and comments, and thank Michael Beasleyand Jonathan Toomer for invaluable assistance in data preparation.

References1 Savioli, L. et al. (1997) Control of schistosoniiasis Ð a global

picture. Parasitol. Today 13, 444Ð4482 Warren, K.S. et al. (1993) Helminth infections, in Disease Control

Priorities in Developing Countries (Jamison, D.T. et al., eds), pp131Ð160, Oxford University Press

3 Bundy, D.A.P et al. Intestinal nematode infections, in HealthPriorities and Burden of Disease Analysis: Methods and Applicationsfrom Global, National and Sub-national Studies (Murray, C.J.L. andLopez, A.D., eds), Harvard University Press for the WHO and theWorld Bank (in press)

4 Snow, R.W. et al (1999) A preliminary continental risk map formalaria mortality among African children. Parasitol. Today 15,99Ð104

5 Omumbo, J. et al. (1998) Mapping malaria transmission intensityusing geographical information systems (GIS): an example fromKenya. Ann. Trop. Med. Parasitol. 92, 7Ð21

6 Ngoumou P. et al. (1994) A rapid mapping technique for theprevalence and distribution of onchocerciasis: a Cameroon casestudy. Ann. Trop. Med. Parasitol. 88, 463Ð474

7 Rogers, D.J. and Williams, B.G. (1993) Monitoring trypanosomiasisin space and time. Parasitology 106 (Suppl.), S77ÐS92

8 Wenlock, R.W. (1979) Prevalence of hookworm and S.haematobium in rural Zambia. Trop. Med. Hyg. 29, 415Ð421

9 Ratard, R.C. et al. (1990) Human schistosomiasis in Cameroon. 1.Distribution of schistosomiasis.áAm. J. Trop. Med. Hyg. 6, 561Ð572

10 Ratard, R.C. et al. (1991) Ascariasis and trichuriasis in Cameroon.Trans. R. Soc. Trop. Med. Hyg. 85, 84Ð88

11 Ratard, R.C. et al. (1992) Distribution of hookworm infection inCameroon. Ann. Trop. Med. Parasitol. 86, 413Ð418

12 Brinkman, U.K. et al. (1988) Experiences with mass chemotherapy inthe control of schistosomiasis in Mali. Trop. Med. Parasitol. 39, 167Ð174

13 Taylor, P. and Makura, O. (1985) Prevalence and distribution ofschistosomiasis in Zimbabwe. Ann. Trop. Med. Parasitol. 79, 287Ð299

14 Crompton, D.W.T. and Tulley, J.J. (1987) How much ascariasis isthere in Africa? Parasitol. Today 3, 123Ð127

15 Utroska, J.A. et al. (1989) An Estimate of the Global Needs for Praziquantelwithin Schistosomiasis Control Programmes. Geneva: WHO, Schsito/89

16 Bundy, D.A.P. et al. (1991) The epidemiological implications of amultiple-infection approach to the control of human helminthinfections. Trans. R. Soc. Trop. Med. Hyg. 85, 274Ð276

17 Stoll, N.R. (1947) This wormy world. J. Parasitol. 33, 1Ð1818 Doumenge, J.P. et al. (1987) Atlas of the Global Distribution of

Schistosomiasis. Bordeaux: Universit de Bordeaux19 Yoon, S.S. (1995) Geographical information systems: a new tool in

the Þght against schistosomiasis, in The Added Value of GeographicalInformation Systems in Public and Environmental Health (de Lepper,M.J.C. et al., eds), pp 201Ð213, Academic Publishers and WHO

20 Nuttall, I. et al. (1998). Systemes dÕinformation geographique etlutte contre les maladies tropicales. Med. Trop. 58, 221Ð227

21 Guyatt, H. et al. (1999) Can prevalence of infection in school-agedchildren be used as an index for assessing communityprevalence. Parasitology 118, 257Ð268

22 Brooker, S. et al. practical application of an index to estimate thenumber of cases of intestinal nematode infections and schistosomiasiswithin a country: Cameroon as a case study. Bull. WHO (in press)

23 Thomson, M. et al. (2000) Environmental information for prediction ofepidemics. Parasitol. Today 16, 137Ð138

24 Thomson, M.C. et al. (1997) Mapping malaria risk in Africa Ð whatcan satellite data contribute? Parasitol. Today 8, 313Ð318

Whole-genome methods are changing the scope of biologicalquestions that can be addressed in malaria research. In the richcontext provided by Plasmodium falciparum genome seq-uencing, genetic mapping is a powerful tool for identifyinggenes involved in parasite development, invasion, transmis-sion and drug resistance. The recent development of a high-resolution P. falciparum linkage map consisting of hundredsof microsatellite markers will facilitate an integrated ge-nomic approach to understanding the relationship betweengenetic variations and biological phenotypes. Here, MichaelFerdig and Xin-zhuan Su provide an overview for applyingmicrosatellite markers and genetic maps to gene mapping,parasite typing and studies of parasite population changes.

The burgeoning data set emerging from thePlasmodium falciparum genome sequencing project isproviding a foundation for advances against thishuman malaria parasite. Knowing gene sequences is acrucial step towards understanding the molecularmechanisms underlying such essential biological pro-cesses as development, transmission, disease patho-genesis and drug resistance, as well as identifying newdrug and vaccine targets; however, even the com-pleted genome of the P. falciparum line 3D7 currentlybeing sequenced will not translate easily into a com-prehensive recognition of genes and their roles in para-site biology. The P. falciparum genome sequence is notonly that of the 3D7 isolate, but a collection of variantsequences from all P. falciparum parasites that share acommon genomic theme. Therefore, complementarymethods are required to deÞne relationships betweengenetic variations (ie. sequence differences) and theirconsequent functional effects.

Microsatellite Markers and Genetic Mapping in Plasmodium falciparum

M.T. Ferdig and X-z. Su

Michael Ferdig and Xin-zhuan Su are at the Laboratory ofParasitic Diseases, National Institutes of Health, 4 Center Drive0425, Bethesda, MD 20892-0425, USA. Tel: +1 301 496 4023,Fax: +1 301 402 0079, e-mail: xsu@helix.nih.gov

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Genetic mapping can be used topinpoint DNA sequences that con-tribute to heritable phenotypes in or-ganisms that undergo sexual recombi-nation. This approach uses high-resolution linkage maps comprisingpolymorphic markers that can bescored easily at hundreds of loci inmany individuals to deÞne a uniquepattern of inherited alleles, known as ahaplotype, for each parasite clone. Therelationship between inherited geneticvariants (speciÞc marker alleles) andphenotypes can be used to identifychromosomal regions that harborgenes affecting traits in either inbredcrosses or outbred populations. Incontrast to biochemical approaches ofworking through proteins and knownpathways to gene discovery, geneticmapping uses phenotypeÐgenotypeassociations to uncover markerslinked to diseases or other biologicalprocesses. This purely genetic ap-proach requires no a priori knowledgeof the biochemical events that give riseto a trait, although it is possible to usegenetics to test a hypothesis about therole of a speciÞc gene in conferring aphenotype.

To this end, a recently developed,high-resolution genetic linkage mapconsisting of .800 polymorphic microsatellite (MS) markers (MalariaGenetics and Genomics on WorldWide Web at http://www.ncbi.nlm.nih.gov/Malaria/index.html)1 (Table 1)will interact coherently with the

Table 1. MS markers and recombination parameters of the Plasmodium falciparum linkage mapa

Physical Genetic Uniquely Physical Genetic SequenceLinkage Total MS RFLP length length mapped resolution resolution statusgroup markers markers markers (Mb)b (cM) segments (kb/marker) (cM)c Kb/cM (center)d

1 27 26 1 0.75 37.5 10 27.8 3.8 20.0 Closure (S)2 41 39 2 0.95 60.4 15 23.1 4.0 15.7 Finished (T)3 67 64 3 1.06 74.8 20 15.8 3.8 14.2 Finished (S)4 61 56 5 1.52 80.5 22 24.9 3.7 18.9 Closure (S)5 62 60 2 1.6 91.8 20 25.8 4.6 17.4 Shotgun (S)6 50 47 3 1.65 106.3 23 33.0 4.6 15.5 Library (S)7 66 54 12 1.44 80.4 16 21.8 5.0 17.9 Library (S)8 32 26 6 1.72 105.2 18 53.8 5.8 16.3 Library (S)9 85 83 2 1.78 106.4 24 20.9 4.4 16.7 Shotgun (S)

10 53 46 7 2.1 94.6 19 39.6 5.0 22.2 Shotgun (T)11 49 37 12 2.4 157.5 22 49.0 7.2 15.2 Closure (T)12 84 75 9 2.7 160.7 35 32.1 4.6 16.8 Closure (St)13 116 111 5 3.2 184.5 40 27.6 4.6 17.3 Closure (S)14 108 101 7 3.4 215.4 42 31.5 5.1 15.8 Closure (T)

Wholegenome 901 825 76 26.3 1556 326 29.2 4.7 16.9

a Abbreviations: MS, microsatellite; RFLP, restriction fragment length polymorphism.b Physical lengths derive from pulsed-field gel data11,29, and from completed chromosome 2 and 3 sequences of the 3D7 strain5,6.c Genetic resolution is defined as the genetic length of the linkage group divided by the number of uniquely mapped segments in the group. Maximum

genetic resolution in 35 progeny is 2.9 cM, directly reflecting recombinational distance (ie. one crossover in 35 progeny is 2.9% recombination).d The task of sequencing the Plasmodium falciparum genome is distributed among three centers: The Sanger Centre (S); The Institute of Genome Research

(TIGR) (T); and Stanford University (St). Status is as of November 15, 1999.

Fig. 1. Diagram illustrating the principle of microsatellite analysis. Parasite isolates A and B have different numbers of the repeat unit TA but identical flanking DNA sequences. PCR primers designed for the flanking sequences are used to amplify the variable repeat region from both parasite DNAs. The PCR product from isolateA is four base pairs longer than the product from isolate B and migrates more slowly in a polyacrylamide gel. The PCR products are labeled with either radioactiveP32 or fluorescent dUTPs and can be detected by autoradiography or laser beam(ABI377), respectively.

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Parasitology Today, vol. 16, no. 7, 2000 309

ongoing genome sequencing effort to drive a Ôpositional candidateÕ approach to identifying genes.In addition, these generally neutral markers provide ameans to discern unique haplotypes that will be essen-tial for characterizing genetic structure and gene ßowin natural populations.

MicrosatellitesMS markers are simple sequence repeats that have

been found in every eukaryote studied2, although theirdensity varies among species. Markers generated fromthese repeats are often polymorphic among parasiteisolates because of the variation in the lengths of theserepeats (Fig. 1). Consequently, MS markers exhibitmultiple alleles in general populations and are very informative for genetic studies. The advantages of MS markers include their general ubiquitousness andthe ease with which they can be scored. MS typing isbased on PCR and requires only a small amount ofDNA, an important feature for studies in which only alimited amount of sample DNA is available. Moreover,the process of genotyping can be automated, and mul-tiple markers can be assayed by multiplexing PCR reactions and labeling PCR products with different ßuorescent dyes.

Although MS markers have been used for geneticstudies in humans and other organisms for more thana decade, P. falciparum is the only Plasmodium speciesfrom which MS markers have been developed. In 1992,Van Belkum et al. reported the presence of mini- andmicrosatellites in several rodent malaria species, butconcluded that there are limited MS markers in thoseparasites3. In contrast to rodent malaria parasites, theP. falciparum genome is rich with microsatellites,mostly (TA)n or (T or A)n (where n is typically between10 and 30), occurring at a frequency of about one perkilobase genome-wide4Ð6. The abundance of MS mark-ers in the P. falciparum genome has facilitated the de-velopment of the linkage map, laying the foundationfor genetic characterization of P. falciparum parasites.

Trait mappingMapping trait loci using genetic crosses. Meiotic recom-

bination occurs in the sexual phase of the life cycle of themalaria parasite in the mosquito host. This shuffling ofchromosomes and chromosomal segments via assort-ment and homologous recombination provides the op-portunity to use genetic linkage to identify genomic re-gions controlling important biological traits. Mapping of such traits depends on the segregation relationshipsbetween markers and phenotypes in the progeny of alaboratory-generated cross of genetically and phenotyp-ically distinct parental lines. The primary considerationin choosing parents for a laboratory cross is that they dif-fer for the phenotype of interest and that the phenotypeis largely heritable (ie. maximally reßects the underlyinggenotype, with little or no environmental inßuence).Other factors dictating the ease with which trait-confer-ring loci can be deÞned are the number of genes involvedand the relative magnitudes of their effects. For example,if a trait is fully heritable and a single gene controls mostof the phenotypic variation in the progeny, then poly-morphic markers tightly linked to a genomic segmentcarrying that gene will co-segregate exactly with the phe-notype, thereby pinpointing a crossover-deÞned intervalthat carries a gene controlling the inheritance of the trait.

The chloroquine response in P. falciparum is an ex-ample of a trait that is primarily determined by a sin-gle major genetic effect segregating in the Dd2 3 HB3cross. The determinant involved in chloroquine resis-tance (CQR) was initially mapped to a 400 kb DNAlocus on chromosome 7 using 16 progeny from thecross and 88 restriction fragment length polymor-phism (RFLP) markers7. Further development of MSmarkers and isolation of more than 1000 additionalprogeny identiÞed new crossovers in this region,thereby narrowing the locus to 36 kb and leading to theidentiÞcation of CQR candidate genes8. Other exam-ples of P. falciparum traits that have been mapped are a defect in male gametocyte development9, folatemetabolism10, invasion and growth rate11 and responsesto several antimalarial compounds. An importantmalaria model, P. chabaudi, has also been used in map-ping studies to identify loci contributing to CQR12.

Most traits are complex, involving the effects ofmultiple genes variously interacting with each otherand the environment. Resolution of these multigenic,complex traits into discrete quantitative trait loci (QTL)in P. falciparum has been made possible by the devel-opment of the high-resolution linkage map. QTL map-ping procedures test the likelihood that each geneticinterval, genome-wide, contains a gene or genes thatcan account for some of the phenotypic variation in asegregating population. Once the phenotypes andgenotypes of a large number of progeny are scored, itis possible to dissect out genetic determinants13. Thenumber of progeny required to achieve the necessarystatistical power to deÞne loci will depend on the de-gree of genetic complexity of a phenotype.

The process of trait mapping and, ultimately, geneidentiÞcation, will be dramatically facilitated by theongoing P. falciparum genome-sequencing project.Each MS marker in the linkage map corresponds to asequence-tagged site (STS) that can be readily identi-Þed in the expanding sequence contigs. Consequently,mapped loci can be tied to physical sequences, and spe-ciÞc candidate genes residing in these sequences can beidentiÞed and evaluated for their possible roles in traitdetermination. In addition, the sequencing project willidentify thousands of simple sequence repeats fromwhich new markers can be developed for saturation ofgenetically interesting regions. The once difficult, long-term task of positional cloning will be condensed to a bioinformatics problem; however, proof of the role of any candidate gene will continue to be a rigorous experimental process.

Allelic association. Although trait mapping using lab-oratory crosses is an important tool for studying the in-heritance of malaria phenotypes, there are limitationsto this approach. It is expensive and laborious to gen-erate laboratory crosses; to date, there are only two P.falciparum crosses available7,14. Moreover, the advan-tages of using laboratory crosses as vehicles for estab-lishing alleleÐphenotype relationships are also part oftheir weakness: the inßuences of genetic backgroundsfrom the parents chosen for a cross are ÔÞxedÕ andmight not be representative of gene interactions thatcould be crucial to the determination of a trait in allel-ically diverse natural populations.

Because genetic recombination of malaria parasitestakes place frequently in the Þeld, trait mapping bygenome-wide allelic association (GAA) in populations

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310 Parasitology Today, vol. 16, no. 7, 2000

is a complementary alternative to studies based onlaboratory crosses. This type of study differs from controlled crosses by evaluating the differences in frequency of genetic variants (alleles) between populations of unrelated ÔaffectedÕ individuals andcontrols. The approach relies on an association,known as linkage disequilibrium, between a variablephenotype and neutral markers located near a gene essential for the determination of that phenotype.Effective GAA mapping is particularly dependent on a genetic map with a high-density marker set, and beneÞts greatly from high-throughput genotyp-ing technologies15,16. A haplotype is generated for each parasite isolate by scoring hundreds of locithroughout the genome. These unique haplotypes,along with precise phenotypic measures, are the rawmaterials for GAA.

Several factors can affect the degree of linkage dis-equilibrium, which, in turn, affects the chance of iden-tifying an association between genetic markers andvariant genes17,18. The primary parameters for increas-ing the chance of detecting linkage disequilibrium aremany well-dispersed markers and large sample sizes.For example, because of the unusual genome-wideuniformity of the physical to genetic distance relation-ship in the P. falciparum genome1, the physical distanceseparating a marker from a genetic determinant will ef-fectively deÞne the chance that a recombination eventwill occur between them; consequently, a high-densitymarker set will be more effective in identifying associ-ations. The number of parasite generations since theoriginal mutation is also an important factor, becauseit determines the number of opportunities for meioticrecombination to disrupt linkage disequilibrium. Thetransmission rate is, therefore, a crucial parameter in P.falciparum studies, because passage of malaria para-sites through mosquitoes is the only opportunity for

meiotic recombination. The higherthe transmission rate and older themutations, the less the chance of detecting linkage between markersand affected genes. Two recent stud-ies on the P. falciparum PfMSP1(Plasmodium falciparum merozoitesurface protein 1) gene illustrate thispoint. In one study using samplescollected from regions with hightransmission, linkage disequilib-rium could not be detected betweenmarkers located more than 0.3 kbapart in the PfMSP1 gene19; how-ever, in a similar study, Sakihama et al. found that linkage disequilib-rium was maintained in the 59 and 39regions of PfMSP1 in samples col-lected from a lower transmission re-gion20. Although explanations suchas functional constraints or balanc-ing selection, speciÞc to a geograph-ical region, can be invoked, one parsimonious explanation is thatlinkage disequilibrium has beenmaintained as a result of limitedmeioses in the lower transmissionregion. Finally, the degree to whichsexual recombination can disrupt

linkage disequilibrium in P. falciparum is affected bythe chance of a mosquito ingesting genetically distinctparasites in the same bloodmeal because recombina-tion can only be ÔobservedÕ as a result of mixed infec-tions. Therefore, the allelic complexity of a local para-site population has an important impact on thedetection of linkage disequilibrium.

Mutations contributing to drug resistance in P. falci-parum provide a unique opportunity to detect allelicassociations, because most of the mutations haveoccurred within the past 50 years, in contrast to poly-morphisms in genes such as PfMSP1 that could haveaccumulated over thousands of years. For example,parasites resistant to chloroquine are thought to haveoriginated from two foci in South Asia and SouthAmerica in the early 1960s and spread to the Africancontinent in the early 1980s from South Asia, as late asthe 1990s in some regions 8,21. Linkage disequilibriumin the 36 kb DNA segment on chromosome 7 that carries a determinant for CQR has been demonstratedamong isolates from South Asia and Africa8. Each ofthe CQR isolates reported in this study was found tocarry the same haplotype in this segment. A survey ofparasites collected from travelers returning fromAfrica also showed 84Ð96% linkage of K and v repeatsin the cg2 gene to the CQR phenotype22. One expla-nation for this association is that the variants confer-ring CQR are relatively recent founder mutations;however, a selected interaction (ie. epistasis) betweenmultiple mutations in the same or different genes isalso a possibility. Drug selection could play a role inmaintaining the haplotype in this locus, because chloro-quine-sensitive (CQS) parasites in a patient containingmultiple infections would be killed by drug treatment,thereby eliminating the chances of recombinationbetween CQR and CQS parasites in mosquitoes. Thesedata argue that GAA mapping using a high density of

Fig. 2. Microsatellite typing of Plasmodium falciparum parasites. Six microsatellite (MS)markers from different chromosomes were typed on DNA from 12 field isolates.Unique patterns could be established for each field isolate. G, green; Y, yellow; B, blue.Note that for some MS markers, two bands are present because of different mobilityof the opposite DNA strands in polyacrylamide gels.

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markers is practical for mapping Plasmodium traitsaffected by recent mutations, such as drug resistance,especially when DNA samples are collected from low-transmission areas or from regions of ÔspreadingfrontlinesÕ of the resistant parasites.

Population studies and parasite typingApplying large numbers of MS markers to Þeld

samples will also reveal important information onparasite population structures and transmissiondynamics. With the completion of the genomesequence of the 3D7 line, the next phase of genome-wide characterization will include the generation ofdatabases of cataloged variations within and amongparasite populations for a better understanding ofparasite evolution, transmission dynamics and theinteractions of these variations with the more com-plex human genome sequences.

Most existing studies of parasite populations haveemployed a limited number of markers from the genesencoding parasite surface molecules such as MSP-1,MSP-2 and circumsporozoite protein (CSP)23. Polymor-phisms among this limited number of genes cannotfaithfully reßect the global relationships of parasite iso-lates and their genome variations. Population studiesusing markers derived from genes that encode theseantigens, rather than generally neutral MS markersderived from non-coding regions, could be inßuencedby host immune pressure. For example, these genesmight be more diverse than general sequences as aresult of immune pressure for antigenic variants, givingrise to an over-interpretation of variability. The appli-cation of a large number of MS markers to Þeld sam-ples would be expected to yield less-biased, in-depthinformation on parasite transmission and populationstructure. In fact, important population parametershave been generated from parasites collected fromPapua New Guinea using MS markers24. The dynamicsof parasite populations in a local area over several yearscan be tracked with a set of MS markers, allowing forthe study of patterns of parasite population changesand gene ßow.

MS markers are excellent tools for parasite identiÞ-cation in laboratory cultures and for ÞngerprintingÞeld isolates. PCR-based typing methods, designed toamplify across repetitive elements in the genome, havegreatly improved the efficiency of parasite identiÞ-cation and are useful for the veriÞcation of culturedparasite lines25Ð27; however, parasite typing using mul-ticopy elements cannot distinguish multiple infectionsin samples collected from patients. Genome typingusing a combination of MS markers can overcome thisproblem, because most of the MS markers are singlecopy and produce only a single band (Fig. 2). Morethan one band will be detected if mixed infections withdifferent alleles are present in a blood sample. A com-bination of 10Ð20 single-copy MS markers will provideenough information to distinguish parasites withrelated genetic backgrounds and to verify the identityof parasite isolates. Ten unlinked MS markers wereused to identify unique recombinant progeny from theDd2 3 HB3 cross.

Genome sequence assemblyGenetic maps are useful not only for mapping

traits, but also for the assembly of DNA sequences.

MS markers (deÞned by a pair of unique primersequences) ordered in linkage groups can function aslandmarks for anchoring DNA sequences into dis-crete genetic regions for efficient local assembly andfor aligning and orienting larger contigs.

MS markers and chromosomal maps are particu-larly useful for the assembly of P. falciparum DNAsegments, because the genome of this parasite isextremely AT-rich, and initial DNA contigs fromshotgun sequencing are typically small owing to diffi-culties in sequencing AT-rich DNA sequences. Toconnect gaps between the DNA contigs, physicalmaps constructed with yeast artiÞcial chromosomes(YACs) have been used to guide the assembly. Unfor-tunately, many P. falciparum chromosomes are notpresently covered by YACs; furthermore, chimericYAC molecules have confounded the assemblyprocess. The genetic map, together with a recentlydescribed optical map28, provides additional means toguide DNA sequence assembly. Because microsatel-lites are so abundant in the P. falciparum genome,more markers can be developed from shotgunsequences and placed in chromosomal linkage maps,leading to the Þnal placement of the DNA contigs ontheir chromosomes.

Future prospectsWith the development of high-resolution MS maps

and advances in technologies to map P. falciparumgenes, it becomes crucial to identify and measurephenotypes for mapping studies. In addition to suchobvious traits as drug resistance, parasite develop-ment and virulence, more subtle phenotypes also willbe segregating. For example, the parasite growth ratecan be divided into variations in the parasite growthcycle and invasion efficiency. Virulence can be repre-sented by the combined effects of toxic moleculesreleased by the parasites and parasite moleculesinvolved in immune evasion mechanisms. Parasitemutants can be generated through in vitro mutagene-sis or drug selection and used in laboratory crosses.Careful observation and deÞnition of phenotypesfrom the Plasmodium parasites will become an impor-tant step in future genetic studies of malaria parasites.

AcknowledgementsWe thank Pradipsinh Rathod, Bruce Christensen, and David Severson for helpful discussions in developing this manuscript;Thomas Wellems for valuable discussion; and Brenda R. Marshallfor editorial assistance.

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3 Van Belkum, A. et al. (1992) Mini- and micro-satellites in thegenome of rodent malaria parasites. Gene 118, 81Ð86

4 Su, X-z. and Wellems, T.E. (1996) Toward a high-resolutionPlasmodium falciparum linkage map: polymorphic markers from hundreds of simple sequence repeats. Genomics 33,430Ð444

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24 Anderson, T.J.C. et al. (1999) Twelve microsatellite markers forcharacterization of Plasmodium falciparum from Þnger prick bloodsamples. Parasitology 119, 113Ð125

25 Carcy, B. (1995) Plasmodium falciparum: typing of malariaparasites based on polymorphism of a novel multigene family.Exp. Parasitol. 80, 463Ð472

26 Su, X-z. and Wellems, T.E. (1997) Plasmodium falciparum: a rapidDNA Þngerprinting method using microsatellite sequenceswithin var clusters. Exp. Parasitol. 86, 235Ð236

27 Su, X-z. et al. (1998) Plasmodium falciparum: parasite typing by using a multiple microsatellite marker, pfRRM. Exp. Parasitol. 89,262Ð265

28 Lai, Z. et al. (1999) A shotgun optical map of the entire Plasmodiumfalciparum genome. Nat. Genet. 23, 309Ð313

29 Gu, H. et al. (1990) Plasmodium falciparum: analysis ofchromosomes separated by contour-clamped homogenouselectric Þelds. Exp. Parasitol. 71, 189Ð198

In their Comment article (this issue), Bundy et al. point out that our studies1 on Ethiopianimmigrants to Israel have an inherentlimitation because we deal with populationsafflicted with health problems other thanhelminthic infections alone. Furthermore,Bundy et al. suggest that the normalization ofthe T-helper type 1/T-helper type 2(Th1/Th2) profile and the decreased immuneactivation that we observed in the Ethiopiansfollowing their immigration to Israel2–5

may reflect changes associated withimmigration, eg. nutrition and better hygiene,rather than reflecting the effect ofdeworming.

We now have additional evidence thatlends support to our original findings andwhich, in part, answers these comments (Z. Bentwich, unpublished). (1) We havecarried out an additional study comparing 30Ethiopian immigrants who receivedantihelmintic treatment, with a group of 19immigrants who unintentionally did notreceive such treatment, a year followingarrival of both groups in Israel. In theuntreated group, despite changes in nutrition,hygiene, etc., immune activation, decreased

CD41 counts and dominant Th2 profilespersisted. In the treated group, thesederangements returned almost to the controllevels (in healthy non-Ethiopian Israeliindividuals)2–5, which we consider normal.The only apparent difference between thetwo groups remained the persistence of thehelminthic infection in the untreated group.This strongly suggests that immune activationis the result of the helminthic infection alone,and that eradication of helminths results inthe amelioration of the immune activation.(2) In another study carried out in Ethiopiaon HIV-infected Ethiopians, with or withoutconcomitant helminthic infections (D. Wolday, unpublished), two majorobservations were made: (i) there was asignificant correlation between the numberof excreted worm eggs and plasma HIV viralload; and (ii) after antihelminthic treatment(given to all participants of the study), therewas a significant relative reduction of HIVplasma viral load in individuals in whomhelminthic infections were eradicated, incomparison to those in whom helminthicinfections persisted or were not present atall. Thus, within the same Ethiopian

environment, helminthic infection increasesHIV viral load, which would lead to fasterprogression of HIV infection.

Taken together, these results support ourbasic premise that eradication of helminthicinfection by itself may have a major impacton AIDS and TB epidemics.

References1 Bentwich, Z. et al. (1999) Can eradication of

helminthic infections change the face of AIDSand tuberculosis? Immunol. Today 20, 485–487

2 Bentwich, Z. et al. (1996) Immunedysregulation in Ethiopian immigrants in Israel:relevance to helminth infections? Clin. Exp.Immunol. 103, 239–243

3 Kalinkovich, A. et al. (1998) Decreased CD4and decreased CD8 counts with T-cellactivation is associated with chronic helminthinfection. Clin. Exp. Immunol. 114, 414–421

4 Bentwich, Z. et al. (1997) Pathogenesis ofAIDS in Africa: lessons from the Ethiopianimmigrants in Israel. Immunologist 5, 21–26

5 Weisman, Z. (1999) Infection by different HIV-1subtype (B and C) results in a similar immuneactivation profile, despite distinct immunebackgrounds. J. Acquired Immune Defic. Syndr.Hum. Retrovirol. 21, 157–163

Zvi BentwichR. Ben Ari Institute of Clinical Immunology and Aids Center

Kaplan Medical CenterHebrew University Hadassah Medical SchoolRehovot, 76100 Israel

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