DIFFERENT GENETIC CHARACTERISTICS OF PLASMODIUM FALCZPARUM ISOLATES COLLECTED DURING SUCCESSIVE CLINICAL MALARIA EPISODES IN SENEGALESE CHILDREN

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1 Am. J. Trp. Med. Hyg., 5(6), 1996, pp Copyright Q 1996 by The Americn Society of Tropicl Medicine nd Hygiene ' DIFFERENT GENETIC CHARACTERISTICS F PLASMDIUM FALCZPARUM ISLATES CLLECTED DURING SUCCESSIVE CLINICAL MALARIA EPISDES IN SENEGALESE CHILDREN AND DILE MERCEEAU-PUUALN Unite d'immunologie Moleculire des Prsites, Institut Psteur, Pris, Frnce; Utiite d'epideniiologie, Institut Psteur, Dkr, Senegl: Lbortoire de Pludologie, Institut Frncis de Reckerclie Scientijque pour le Developpement en Coopertion (RSTM), Dkr, Senegl Abstrct. A nrrow epidemiologic survey ws conducted during four-month period of intense mlri trnsmission in Dielmo, holoendemic Seneglese villge. Longitudinl clinicl nd prsitologic follow-up indicte tht clinicl mlri episodes lwys occurred fter n brupt increse in prsite densities. Polymerse chin rection nlysis of Plsmodium flciflruin prsites ws crried out in blood smples collected longitudinlly from 10 children who hd experienced severl clinicl episodes during this period. ur dt show tht the genetic diversity of the prsites circulting in this villge is very lrge. The successive clinicl episodes experienced by ech child were cused by geneticlly distinct prsite popultions tht were recently inoculted nd multiplied in n pprently unrestricted mnner. Importntly, the genetic chrcteristics of the prsite popultions detected during phses of symptomtic crrige differed from those cusing clinicl episode, suggesting tht the vrious fctors tht control of prsite growth in these children re strin-specific. In nive individuls, infection by mlri prsites lmost invribly results in clinicl episode. In humns living in regions where mlri trnsmission is high, clinicl mlri primrily ffects young children nd the presence of bloodstge prsites is not synonymous with disese. A significnt proportion of young children, most older children, nd mny dults crry prsites without presenting clinicl symptoms.' It is generlly dmitted tht this reflects the efficiency of the progressively cquired mlri-specific immunity, which prevents clinicl mnifesttions nd reduces prsite burden. To dte, little is known bout how this protective immunity is elicited, how it opertes, nd how prsitiztion results in disese?. Experimentl infections in humns with defined prsite strins hve shown tht n efficient strin-specific immunity ws cquired fter infection with one strin, llowing the individul to resist infection by tht strin but providing little protection ginst heterologous Severl infections with the sme strin were, however, required to elicit sterile immunity to tht trin.^ These findings showed tht both poor immunogenicity nd strin diversity my represent serious obstcles to the development of protective immunity. The relevnce of these observtions to the sitution fced by humns living in mlri-endemic regions is still obscure. While there is now mple evidence tht mlri prsite species re polymorphic for lrge number of chrcteristics,6 the extent of genetic diversity of locl prsite popultions to which people re ctully exposed is essentilly unknown, nd virtully nothing is known bout the circultion of strins in restricted geogrphic re.7.8 Therefore, the question of whether prsite diversity plys mjor role in the occurrence of clinicl episodes in endemic regions, key element in our understnding of hosdprsite reltionships, is still unnswered. To ddress this issue, we hve crried out n nlysis of the genetic chrcteristics of Plsinodiuiiz flci~>ruin prsites collected longitudinlly from children who experienced severl successive clinicl episodes. In Dielmo, Seneglese villge where mlri is holoendemic, close follow-up of the entire popultion of the villge ws crried out during the 1990 riny se~on.~ As result, longitudinl records of prsite densities of P. fkiprum, P. mlrie, nd P. ovle nd of clinicl events re vilble for ech inhbitnt of the villge during this four-month period of high trnsmi~sion.~ The men inocultion rte ws pproximtely one P. flciprum infective bite every second night nd most children 1-6 yers of ge experienced more thn one clinicl mlri episode. During this survey, cpillry blood smples were collected t two-week intervls s well s during clinicl episodes, llowing longitudinl nlysis of the chrcteristics of the prsites infecting the inhbitnts of this villge over four-month period of intense mlri trnsmission. Plsmodium flcipruin prsites circulting in humn peripherl blood re hploid ring stges. We hve crried out typing procedure bsed on the polymerse chin rection (PCR) tht uses DNA from circulting prsites to nlyze severl single-copy genetic loci. The lrge llelic diversity reported in regions such s block 2 of the merozoite surfce ntigen-1 (MSA-1) gene or the centrl repetitive domin of the MSA-2 gene, differing both in nucleotide sequence nd in number of repetitive sequences, or in the thrombospondin relted nonymous protein (TRAP) gene, presenting restriction site polymorphism, renders these mrkers prticulrly convenient for typing purposes.10 Distinct prsite popultions cn redily be identified using one or combintion of these chrcters.i0. II Such typing pproch ws used in the work reported here to nlyze the chrcteristics of P. fkiprum prsites collected from 10 children who hd experienced two or more clinicl mlri episodes during the four-month period of intense follow-up in Dielmo. MATERIALS AND METHDS Study site. The Dielmo villge is locted in the Ftick region, 280 km southest of Dkr, Senegl. After reching

2 TYPING F SUCCESSIVE CLINICAL MALARIA ISLATES 6 n greement with the leders nd the popultion of the villge, s well s with the ntionl uthorities, n epidemiologic study ws undertken in June A detiled description of Dielmo nd of the epidemiologic study conducted in this villge hs been reported el~ewhere.~ Due to the presence of strem, mlri trnsmission is perennil. From June 1 to September 0, 1990, when the smples nlyzed were collected, the inocultion rte ws pproximtely one infective P. flciprum bite every second night. Longitudinl clinicl nd prsitologic follow-up. The clinicl, prsitologic informtion reported here for 10 children ws collected during the close follow-up of the entire popultion of the villge (27 inhbitnts) crried out from June 1 to September 0, Briefly, ech person ws visited t home three times week to record temperture (rectl temperture for the children less thn seven yers of ge) nd to dminister clinicl questionnire concerning the pst 8 hr. In ddition, ech compound ws visited dily to rpidly detect new cses of fever. Twice week, thick blood smer ws mde from ech person in the villge. In l1 ptients with fever, dditionl blood smers were tken. The thick blood smers were stined with Giems nd t lest 200 oil-immersion fields were exmined by microscopy. Prsites were quntified s described by Trpe nd other^.^.^^ Since prsite prevlence in the villge ws very high, cutoff in prsite density ws used to define mlri episodes nd decide upon tretment. For children, clinicl mlri episode ws defined s fever (rectl temperture 2 8.5"C) ssocited with prsite:leukocyte rtio 2 2, i.e., 2 16,000 prsites/p1.12 Tretment consisted in dministrtion of Quinimx@ (Snofi-Lbz, Pris, Frnce) t dose of 25 mg/kg/ dy in three dily doses over three dys. As prt of the protocol, the Dielmo villgers were sked not to use chemoprophylxis or utomediction without informing the tem. Use of ntimlrils by the popultion ws indeed restricted to our prescriptibns, s indicted by questionnire nd monthly urine tests.9 Collection of blood smples. Blood smples were collected twice month using cpillry pipettes nd venous blood smples were collected during mlri ttcks. The smples were centrifuged, the buffy cot ws discrded, nd the red blood cell pellet ws frozen in liquid nifrogen in the villge. After trnsporttion, the tubes were stored t -80 C. The protocol ws pproved by the Ministere de l Coopertion et du Developpement nd the Ministere de l Snte Publique of Senegl. IFformed consent ws obtined individully from the prticipnts or their prents. Extrction of DNA. Using stndrd procedures,1 DNA ws extrcted. Briefly, pproximtely 100 pl of red blood cells were lysed by three freeze-thw cycles. Free prsites were wshed three times with distilled wter nd resuspended in five volumes of TEN buffer (10 mm Tris-HC1, ph 8.0, 1 mm EDTA, 0.15 M NCl), 0.5% Triton X 100, 0.5% sodium dodecyl sulfte, nd 5 mg/ml of proteinse K. After l hr of incubtion t 7"C, the DNA ws extrcted twice with one volume of phenolkhloroforrdisomyl lcohol (25/2/1), nd precipitted with ethnol in the presence of 0. M sodium cette (ph 5.5). The DNA ws resuspended in 50 p1 of wter. Amplifiction by PCR. The PCR procedures, permitting nlysis of polymorphic regions of the MSA-1, MSA-2, nd TRAP loci, re detiled elsewhere.1 Briefly, mplifiction ws done using 1-5 pl of DNA in finl volume of 50 pl in the presence of 200 pm of ech dntp, 1 pm of ech primer, nd 2.5 U of Tq I DNA polymerse (Promeg Frnce, Chrbonnieres, Frnce) in the buffer supplied by Promeg (12 mm MgCl,). The rections were performed in Hybid thermocycler (Cer-Lbo, Aubervilliers, Frnce). The stndrd rections prmeters were 15 sec t 9 C 2 min t 55"C, 2 min t 72 C (one cycle), 2 sec t 9"C, 2 min t 55"C, 2 min t 72 C (5 cycles), nd 2 sec t 9"C, 2 min t SC, nd 10 min t 72 C (one cycle). The primers used for MSA-1 were MSA-1 A: 5'AAG CTT TAG AAG ATG CAG TAT TGA C' nd MSA-1 B: 5'ATT CAT TAA TTT CTT CAT ATC CAT C'. For the MSA-2 gene the following primers were used: MSA-2 1: 5'ATG AAG GTA ATT AAA ACA TTG TCT ATT ATA' nd MSA-2 2: 5'AAC GAA TTC ATA AAC AAT GCT TAT AAT ATG AGT'; MSA-2 : 5'GAT GAA TTC TAG AAC CAT GCA TAT GTC CAT GTT' nd MSA-2 : 5'ATA TGG CAA AAG ATA AAA CAA GTG TTG CTG'. For the mplifiction of the TRAP gene, the following primers were used: TRAP : 5'ATG TAA CTT GTA TGC TGA TTC TGC ATG G' nd TRAP : 5'TAT CTT CAC TAT TAG GTA CGT GCC TAT TTC C'.lo Anlysis of PCR products nd llele ssignment. The PCR products were nlyzed for size polymorphism on grose gels (Sekem GTG-Tebu Frnce, Le Penry en Yvelines, Frnce) s described.lo The TRAP lleles were identified by restriction frgment length polymorphism (FWLP) fter digestion with Tq I restriction endonuclese.' The MSA-1 nd MSA-2 lleles were ssigned to llelic fmilies by hybridiztion using fmily-specific probes, s described.lo Probes were prepred by nick trnsltion (nick trnsltion kit; Boehringer Mnnheim, Meyln, Frnce) using specific frgments mplified from reference clones or isoltes, whose sequence hs been determined or from the recombinnt plsmids obtined fter cloning the vrious MSA-1 nd MSA-2 reference probes (TA cloning kit; In Vitrogen, xon, United Kingdom). A PCR product ws ssigned to specific llelic fmily if it hybridized with the corresponding probe under stringent conditions (0.1X SSC 115 mm NCl, 15 mm sodium citrte, ph 7.01 t 65 C). The frgments tht hybridized to one or more probe t moderte stringency (SX SSC t 65"C), but no longer t high stringency (0.1X SSC t 65 C) were considered hybrids. Frgments tht hybridized to distinct probes under conditions of low stringency (6X SSC t 65"C), produced fint hybridiztion signls t higher stringency (2X SSC t 65"C), nd did not hybridize under more stringent conditions were grouped s unssigned lleles. They most probbly consisted of complex mosic hybrid genes. lo RESULTS A totl of 12 P. flciprurn clinicl mlri episodes were observed ìn the 27 inhbitnts of Dielmo. Most (92.6%) occurred in children less thn 10 yers of ge. Bbies nd children less thn four yers old experienced men of two clinicl ttcks, while older children (5-9 yers of ge) hd men of one clinicl ttck? In the birth to 6- yer-old group, (61%) of 56 children hd more thn one

3 "., I 6 CNTAMIN AND THERS clinicl ttck. None of the children more thn seven yers of ge experienced more thn one ttck.i The longitudinl prsitologic follow-up conducted during this period showed tht mlri ttcks were lwys ssocited with n brupt increse in prsite density. In most cses, drug tretment resulted in rpid resolution of symptoms nd clernce of sexul blood stges in the first few dys. This is illustrted in Figures 1-6, in which the longitudinl records of prsite densities of the children studied re presented. In this work, we hve investigted some genetic chrcteristics of P fkiprum prsites collected longitudinlly from children who hd experienced two or more clinicl episodes during the study period. Ten representtive children, who presented between clinicl episodes blood smers tht were either 1) constntly negtive or 2) constntly positive, but with low prsite densities (trophozoites ndlor gmetocytes), were chosen. A totl of 89 DNA preprtions were mde, including 28 from P. flciprunz clinicl mlri episodes. The PCR typing ws crried out for three distinct genetic loci locted on distinct chromosomes.i0 For MSA-1 (lso clled MSP-I), the highly polymorphic block 2 ws mplified. For MSA-2 (lso clled MSPr2), two independent rections were done, mplifying the entire coding region (primers 1 + ) or the centrl polymorphic domin (primers 2 + ). The TRAP mplifiction concerned the centrl region of the gene. All PCR primers used here were highly specific for P. fkiprum prsites, s tests with other humn mlri species did not yield ny PCR product.io Anlysis of the mplified frgments ws done by investigting size polymorphism in ll cses, nd RFLP ws investigted for the TRAP rection. Both MSA-1 nd MSA-2 frgments were ssigned to specific llelic fmilies (MAD 20, K1, nd R0 for MSA-I nd D7 nd FC27 for MSA-2) by hybridiztion using fmily-specific probes. lo Anlysis of the prsites collected during successive mlri episodes. We first nlyzed the clinicl episodes of children presenting constntly negtive smers between the ttcks. A typicl exmple is illustrted in Figure 1, showing prsite typing from 1.5-yer-old girl, code 02/0, who experienced two clinicl mlri episodes (rbitrrily designted A nd B in Figure l), with high prsite densities, both of which were successfully drug-treted. Aprt from the smers collected during the ttcks or for the following 2- dys, ll thick blood smers collected from this child were negtive. Eight successive blood smples (numbered 1-8) were vilble. The DNAs 1- nd 6-7 were collected s prt of the s'ystemtic survey, nd the corresponding thick blood smers were negtive. The PCR from these smples ws lso negtive for ll mrkers investigted (Figure 1, lnes 1- nd 6-7, respectively), Figure 1 shows tht DNAs nd 5, collected two dys prt during episode A, presented the sme mplifiction ptterns for MSA-1 nd MSA- 2, with two bnds for ech mrker. The pttern obtined for DNA 8, collected during episode, however, produced single bnd of different size for both rections. Assignment to specific llelic fmilies ws done by hybridiztion using fmily-specific probes,io s summrized in Tble 1. The MSA-1 frgments generted from DNAs nd 5 hybridized with the K1 fmily-specific probe, while the frgment obtined from DNA 8 hybridized with the MAD20 fmily- f o A o period of survey A B o + A B 8* FIGURE 1. Course of Plsmodium fkiprum prsitemi (top) nd typing of the prsites (bottom) collected from child 02/0, 1.5-yer-old girl, who experienced two clinicl ttcks, denoted A nd E. Thick blood smers were collected twice week. Positive thick blood smers contined exclusively P. fkiprum trophozoites. Prsite densities re expressed s the number of P. fkiprum trophozoites for 100 leukocytes (i.e., 1150-U100 IJ-I of blood). DNAs were prepred from blood smples collected on June (l), June 22 (2), July 9 (1, July 26 (, episode A), July 28 (S), August 10 (6), August 2 (71, nd September 29 (8, episode B). Clinicl episodes were drug-treted, s described in the Mterils nd.methods. Numbers with n sterisk indicte tht the corresponding thick blood smer ws positive, while for the others, thick blood smers were negtive. Bottom, nlysis of the polymerse chin rection (PCR) products by electrophoresis on grose gels nd stining with ethidium bromide. I, mplifiction of merozoite surfce ntigen- 1 (MSA- 1) using primers A + B. II, mplifiction of MSA-2 using primers 2 + ; III nd IV, typing for thrombospondin relted nonymous protein (TRAP), undigested, nd Tuq I-restricted PCR products, respectively. The numbers bove ech lne refer to the number of the DNA smple nlyzed. = no DNA dded in the rection; + = positive control (DNA from clone 89F5).18 The positions of the moleculr weight mrkers (Boehringer Mnnheim mrker VI) re indicted schemticlly for the 1,0-, 65-, 517-, 5-, 9-, nd 298-bsepir (bp) mrkers in I nd II. The size of the TRAP frgment is indicted in III.,

4 TYPING F SUCCESSIVE CLINICAL MALARIA ISLATES 65 2w01 0/05) P '"1 * -- L A -- - e June July August September period of survey (dys) 0/1) -- b * June - July Augusf period of survey (dys) A 1* B -- r- -- June A -- July August period of survey (dys).) September r- -- r- c June July August Septeniber period of survey (dys) FIGURE 2. Course of Plsmodium flciprum prsitemi in four children. Ech clinicl episode ws drug-treted s described in the Mterils nd Methods. The symbols used re s in Figure 1. 1/05, six-yer-old boy, experienced four clinicl episodes. The child crried exclusively P. flciprum trophozoites. Blood smples were collected on June 20 (1, episode A), July 9 (2), July 11 (, episode B), July 28 (). August 6 (5, episode C), August 20 (6). September 1 (7, episode D), September (8), nd September 17 (9). 19/09, three-yer-old boy, experienced three clinicl episodes. Blood smples were collected on June 2 (I), July (2, episode A), July 7 (), July 21 (). July 25 (5, episode B), August (6), August 18 (7), nd August 19 (8, episode C). The child presented occsionlly, in ddition to the P. flciprum sexul blood stges indicted by n sterisk, low densities of P. fkiprum gmetocytes (smples 1,,, nd 5) nd/or P. mlrie (smples 1 nd 2) prsites during the period under study. 1/1, four-yer-old boy, experienced four clinicl episodes. Blood smples were collected on June (l), June 22 (2, episode A), July 8 (, episode B), July 9 (), July 28 (5, episode C), July 0 (6), August 6 (7), August 20 (8, episode D), September (9), nd September 21 (I). The chiid presented, in ddition to the P. fkiprum trophozoites indicted, low densities of P. mlrie (smples 1, 2, nd ) nd P. ovle (smple 2) prsites. 08/0, five-yer-old boy, experienced two clinicl episodes. Blood smples were collected on June 0 (l), July 10 (2, episode A), July 16 (, episode B), nd July 25 (). The child lso crried low densities of P. flciprum gmetocytes in 11 smples used for DNA extrction. specific probe. Both DNAs nd 5 generted MSA-2 frgments belonging to the FC27 llelic fmily,, nd the single frgment mplified from DNA 8 ws D7-type. TRAP typing (Figure 1, bottom pnel) confirmed tht the prsites from both episodes were geneticlly distinct, since the lleles crried could be differentited by Tq I R EE As shown in Figure 2, three other children (1/05, 19/ 09, nd 1/1) presented negtive smers in the periods seprting two ttcks. Agin, no PCR product ws obtined from the smples with negtive blood smers. Typing of prsites in the smples collected during clinicl episodes is indicted in Tble 1. Child 1/05, six-yer-old boy, experienced four episodes (designted A-D in Figure 2). Typing of these episodes (Tble 1) indicted tht episodes A nd B could be esily distinguished both from ech other, nd from C nd D by their TRAP, MSA-1, nd MSA- 2 mrkers. Prsites C nd D could not be distinguished using MSA-1 nor TRAP, but MSA-2 typing showed tht they were different, since DNA from episode C did not yield ny bnd using primers 2 + nd no signl ws obtined upon hybridiztion. Since this DNA generted PCR product for the other mrkers used, including MSA-2 using primers 1 +, we concluded tht the sequence of the MSA-2 llele from episode C ws such tht primers 2 + did not form stble hybrids. In contrst, mplifiction using primers 2 + ws obtined from DNA collected in

5 66 CNTAMIN AND THERS u u B * c t 5* -- * June -- July August - September period of survey D i'" A B C D A'B c D MSA-1 A+B (I711)+I I KI - I0 MAD20 MS, size (bp) x0 8Rdoublet 65n+50 ollele D7, FC27 FC27 FIGURE. Course of prsitemi nd typing of the prsites from child 1/07, two-yer-old boy, who experienced four clinicl episodes, which were drug-treted s described in the Mterils nd Methods. Tretment of episode A ws incomplete due to vomiting. Blood smples were collected on the dtes indicted. The course of prsitemi is indicted using the sme symbols s in Figure 1. The child crried exclusively Plsmodiuriz flciprurn prsites during this period. Gmetocytes were detected occsionlly; none of the smples used for DNA extrction contined gmetocytes. Results of mplifiction of merozoite surfce ntigen-1 using primers A + B nd MSA- 2 using primers 2 t re shown in I nd II, respectively. Frgment size ws estimted on grose gels, using Boehringer Mnnheim moleculr weight mrker VI. The positions of the mrkers re indicted (from top to bottom: 1,0, 65, 517, 5, 9, nd 298 bsepirs [bp]). The numbers bove ech lne refer to the number of the DNA nlyzed. = no DNA dded in the rection: + = positive control (DNA from clone 89F5).18 The bottom of the figure summrizes genotyping. troph. indictes prsitemi, expressed s the number of trophozoites/100 leukocytes. The ssignment to llelic fmily ws crried out by hybridiztion using fmily-specific probes under conditions of distinct stringency, s described in the Mterils nd Methods. The vrious lleles observed re indicted in the sme order for size nd llelic type. -indictes tht no frgment ws observed, neither by stining with ethidium bromide nor fter hybridiztion. episode D, which lso contined multiple MSA-2 lleles detected by hybridiztion. Child 19/09, three-yer-old boy, experienced three episodes (A-C in Figure 2). During ech, s indicted in Tble 1, the child hd multiple prsite types, yielding three frgments for MSA-1, one or two frgments for MSA-2, depending on the primer combintion used, nd single TRAP frgment. The MSA-1 types of episodes A nd B could not be distinguished, while the lleles present during episode C differed from those detected in the previous ones. Prsites from ech episode hd different TRAP pttern nd specific MSA-2 profile. Child 1/1, four-yer-old boy, experienced four mlri episodes nd hd severl negtive blood smers in the period seprting these episodes (Figure 2). Tble 1 shows tht ech episode presented specific TRAP llele nd unique multiple bnd pttern for both MSA-1 nd MSA-2. ther children presented negtive blood smers shortly fter tretment but could, therefter, hrbor prsites for severl dys or weeks without presenting symptoms before experiencing nother clinicl mlri episode. Typicl exmples re illustrted in Figures -6. Comprison of the genetic chrcteristics of the prsites collected during the successive episodes experienced by these children, indicted in Tble l, gin shows tht the prsites collected during successive clinicl episodes in given child were geneticlly different. Episodes A, B, nd C in child 28/06 differed in their MSA-2 profiles, s did episodes A nd B experienced by child Episodes A nd B in child 12/07 differed in their MSA-2 lleles nd their TRAP profiles. For both episodes in child 22/06, prsites differed t the three loci investigted. There were two cses in which drug tretment did not result in complete clernce of the sexul blood stges within the following few dys. As shown in Figure 2, tretment of episode A experienced by child 08/0 ws followed by 2-fold decrese in prsite density but not by clernce, since prsites were detected in the three blood smers preceding the outgrowth provoking episode B. Typing of the prsites from episodes A (DNA 1) nd B (DNA ) shown in Tble 1 indicted tht for these episodes, the prsites could not be distinguished. Smple DNA 2, collected during the intervl, produced the sme pttern, while DNA, obtined fter drug clernce of pek B, ws negtive. The results of typing re consistent with typicl recrudescence due to incomplete tretment of the first episode. An nlogous sitution ws observed for episodes A nd B of child 1/07, two-yer-old boy. As shown in Figure, prsitemi in this child fluctuted during the month of June, while he remined symptomtic. A rpid increse in prsite density resulted in clinicl episode on June 27 (episode A). Drug tretment of this episode ws incompletely dministered (due to vomiting) nd the child did not cler the prsites. A second clinicl episode (B) ws recorded few dys lter on July. Tretment of this episode ws complete nd the following routine thick blood smers were negtive. As shown in Figure, I nd II, the DNAs collected during episodes A nd B generted the sme 580-bsepir (bp), D7-type MSA-2 llele, nd both generted 70-bp, K1- type MSA-1 llele. Prsites from episode A, however, generted n dditionl MSA-1 bnd, typed s 20-bp

6 1. TYPING F SUCCESSIVE CLINICAL MALARIA ISLATES 67 C 10 * t Y : EI E 00 L Q) E g 200 )NA - dte - roph. 0 size (bp) LA June July b August September period of survey MSA-1 A+B llele size (bp) MSA-2 1+ llele - TRAP profile 1 11/06/ K1, K d7,fc27, FC /06/ K FC27, FC27 -A /07/ K1 80 FC27 1/07/ K1, K1 80 FC27? 5 ~107/ K1, (Kl,MAD20) ,(D7,FC27),(D7,FC27)? 6 6/08/ K1, (KI, MAD20) (D7, FC27), (D7, FC27), FC27 b 7-B /08/90 20/08/90 /09/90 15/09/ K1, (Kl,MAD20), MAD20. - Kl K1, (Kl,MADP), MAD D7, FC27, FC27 FC27 (D7,FC27), FC27 C - d FIGURE. Course of Plusniodiirnt fulcipurum prsitemi nd typing of the prsites from child 28/06, six-yer-old boy, who experienced three clinicl episodes, which were drug-treted s indicted in the Mterils nd Methods. The course of prsitemi is indicted using the sme symbols s in Figure 1. The child presented, in ddition to the P. flcipuncrit sexul blood stges indicted here, low densities of P. fulcipurum gmetocytes (smples nd 8) nd P. muluriue prsites (smples 1, 2,, 7, 8, nd 10) during the period under study. Blood smples were collected on the dtes indicted in the tbulr portion of the figure, which summrizes the results from the typing of the merozoite surfce ntigen-1 (MSA-1) frgments mplified using primers A + B nd of the MSA-2 frgments obtined using primers 2 + nd thrombospondin relted nonymous protein (TRAP). troph. indictes prsitemi, expressed s the number of trophozoitesi100 leukocytes. The size of the frgments ws estimted using Boehringer Mnnheim moleculr weight mrker VI. The llelic type ssigned to the vrious frgments detected is indicted, s deduced from hybridiztion using fmily-specific probes under conditions of distinct stringency, s described in the Mterils nd Methods. The vrious lleles observed re indicted in the sme order for size nd llelic type. Hybrid genes re indicted in prentheses. The TRAP typing ws done using size polymorphism nd Tuq I restriction frgment length polymoxphism. The vríous TRAP profiles hve been ssigned rbitrry codes, -d. bp = bsepirs;? = mbiguous typing results (unssigned llele), - = no frgment observed.

7 68 CNTAMIN AND THERS (o r- b w - June July August Sepleiiibur peried cif survey o o 10 FIGURE 5. Course of Plsinodiunz flciprum prsitemi nd typing of the prsites from child 02/0, four-yer-old girl, who experienced two clinicl episodes tht were drug-treted s described in the Mterils nd Methods. The child crried P. flciprum trophozoites (troph.) nd P. flciprum gmetocytes (dotted line), s indicted. In ddition, low density of P. mlrie prsites ws observed in smple 1. Note tht the scles used for trophozoites nd gmetocytes differ. Nine blood smples, numbered 1-9, were collected on the dtes indicted. For definitions of the symbols used, see Figure 1. Results of mplifiction of merozoite surfce ntigen- 1 (MSA-1) using primers A + B nd MSA-2 using primers 1 t re shown in I nd II, respectively. The numbers bove ech lne refer to the number of the DNA nlyzed. = no DNA dded in the rection; - = positive control (DNA from clone 89F5).18 Genotyping is lso indicted. Prsitemi is expressed s the number of trophozoites/l leukocytes. For size determintion nd llele ssignment, see Figure. MAD20-type, identicl to the one detected from DNA 1, collected during the symptomtic period preceding episode A. While typing indicted tht the prsite popultions present during episodes A nd B were different, the results sug- gested tht in both episodes, the prsites cusing the pthology were those crrying the 70-bp K1 type. This prompted us to nlyze the other symptomtic episodes in the context of the preceding symptomtic crrige. Comprison of prsites crried before nd during clinicl ttcks. The prsites crried by some children in the symptomtic period preceding n ttck were compred with those collected during the clinicl episode. In ddition to episodes A nd B of child 1/07 noted bove, this ws lso crried out for severl episodes in three other children. The prsites from the three episodes experienced by child 28/06 were compred with those collected in the preceding periods. As shown in Figure, DNA 2 yielded two distinct FC27-type MSA-2 frgments of 80 bp nd 780 bp. From DNA, collected five dys lter during episode A, single 80-bp MSA-2 bnd ws generted, suggesting tht the pek prsitemi ws due to multipliction of this prsite type tht becme dominnt. In contrst, DNA 7, collected during episode B, ws more complex thn DNA 6, collected nine dys erlier. The B clinicl ttck ws ssocited with the detection of three MSA-1 nd three MSA-2 frgments. Stining grose gels with ethidium bromide indicted tht this prsite popultion contined mixture of (t lest) three distinct types present in similr proportion. Two MSA-1 lleles ( 60-bp K1-type nd 50-bp, MAD 20- type) mplified from DNA 7 were not observed in the previous smple. Conversely, the 80 bp K1-type llele ohserved in smple 6 ws not detected in DNA 7. Similrly, the 90-bp nd the 870-bp D7iFC27 hybrids mplified from DNA 6 were no longer observed mong the products obtined from DNA 7, while two new lleles, ( 90-bp D7- type nd 820-bp FC27-type), were generted from DNA 7. This indictes tht the prsite popultion collected t the time of the B clinicl ttck contined novel prsite genotypes, but did not contin single dominnt type. Similrly, the ptterns generted from DNA 10, collected during episode c, were more complex thn those of the smple collected 12 dys erlier, t time when the child ws symptomtic (DNA 9). The DNA from episode C generted, in ddition to the single MSA-1 or MSA-2 llele observed in DNA 9, two MSA-1 lleles ( 10-bp KINAD 20 hybrid nd 50-bp MAD20 llele), nd one dditionl 920-bp D7-FC27 hybrid MSA-2 llele. In both rections, however, the intensities of the ethidium bromide stining differed, the prominent bnds being the 60-bp KI MSA-I llele nd the 920-bp MSA-2 hybrid, suggesting tht there ws single dominnt type in DNA 10. Figure 5 shows the course of prsitemi in child 02/0, four-yer-old girl, who experienced two mlri episodes ssocited with symptoms nd high prsitemi, designted A nd B. The other peks of prsitemi were not febrile nd decresed without tretment. The child crried P. flciprurn gmetocytes most of the time, All smples used for DNA preprtion contined gmetocytes, which complicted interprettion of the typing. Results obtined using MSA-1 nd MSA primers re shown in Figure 5, I nd II, respectively. A 80-bp K1-type MSA-1 frgment ws observed in ll smples, including those collected fter Quinimx@ tretment, in which only few gmetocytes nd no sexul forms could be detected. This frgment is, therefore, interpreted s being derived from the sme popultion of,

8 !l TYPING F SUCCESSIVE CLINICAL MALARIA ISLATES 69 z Y Q) I C s \ m s.i 2000 r: 8 k Y Cu PI\ A * (22/06) z I r! - 00 z h u cw d E 1 E: B To I June July August September period of survey - DNA - dte troph size (bp) MSAl A+B llele M size (bp) L-2 1+ llele TRAP profile 1-A 27/06/ MAD D7,D7 2 0/06/ MAD D7,D7 17/07/ MAD20, K1, R0 900 (d7, FC27)? 5 1/07/90 1/08/90 11s * MAD20, K1, R0 (Kl, R0) 900 (d7, FC27) b /08/ (Kl, R0) 900 (d7,fc27) X 7-B 2/09/ (Kl,R0) 900 (d7, FC27) b 8 11/09/ /09/ MAD20, (Kl, R0) 900 (d7,fc27) X FIG& 6. Course of Plusnzodium fulciprum prsitemi nd typing of the prsites from child 22/06, two-yer-old girl, who experienced two clinicl episodes, designted A nd B, which wene drug-treted s described in the Mterils nd Methods. The child crried P.flcipurrtn2 trophozoites nd P. fkiprum gmetocytes (dotted line), s indicted. Note tht the scles used for trophozoites nd gmetocytes differ. For definitions of the symbols used, see Figure 1. Nine 'blood smples, numbered 1-9, were collected on the dtes indicted. Typing results re summrized in the tbulr portion of the figure. troph. refers to the number of trophozoites/l leukocytes. bp = bsepirs. TRAP = thrombospondin relted nonymous protein. For size determintion nd llele ssignment, see Figure. '

9 o\ P -b+-- TABLE 1 Genotyping of Plsmodium fulcipururn prsites from successive clinicl episodes in 10 children* P. fulcipunirti Agc Clinicl tropho- MSA-! A + B MSA-I block 2 MSA MSA-2 TRAP Child (yers) episode Dte zoitest size llelic fmily SlZC llelic fmily profile 02/0 1.5 A 7/26/ K1, K FC27, FC27 1/05 6 B 9/29/90 72.,560 MAD D7 b A 6/20/90 1, x, x FC27,D7 B 7111/90 1,00 10 X. 70 FC27 b 19/09 C 8/6/ KI 920 X C D 9/1/ K1 920 FC27,D7,D7 C A 7//90 1, (KI, MAD20), MAD20,? (D7, FC27), 870 B 7/25/ (KI, MAD20), MAD20,? D7, (D7, FC27) b C 8/19/ ?,?, KI 820 (D7, FC27) C 1/1 A 6/22/ MAD D7 B 7/8/90 1, KI, MAD20, R D7, FC27 b n C 7/28/90 1, K1, MAD20, MAD (D7, FC27) C z D 8/20/ (MAD20, KI) 780 D7 d 28/06 6 A 7// KI 80 FC27 B 8/15/ KI, (KI, MAD20), MAD20 C 9/15/90 1, KI, (Kl, MADZ), MAD20 02/0 A 6/0/90 1,027 12/07 1 A D7, FC27, FC27 'C P (D7, FC27), FC27 d MAD20, KI 680 FC27 NA B 7/0/ MAD20, KI 760 D7 NA s 6/2/ f 00 (MAD20, KI), R0, (MADZ, KI) FC27, FC27 i5 B 7/5/ (MAD20, KI), R0, (MADZ, KI) 80 i- 800 FC27, FC27 b E 22/06 2 A 6/27/90,00 0 MAD D7,D7 B 9/2/ (KI, R0) 900 D7, FC27 b 08/0 5 A 6/0/ (Kl, R0) 880 FC27 B 7116/ (Kl, R0) 880 FC27 1/07 2 A 6/27/ K1, MAD D7 NA B 7//90 1, KI 580 D7 C 7/2/90 1, KI 580 doublet D7, FC27 NA D 9/22/90 1,06 50 MAD FC27, FC27 b *Episodes were rbitrrily coded A-D for ech child. The size of the frgment is expressed in bsepirs. The ssignment to specific llelic fmily ws bsed on the results of hybridiziton using llele-specific probes s described in the Mterils nd Methods. Hybrid genes (intrgenic recombinnts) hybridized with two distinct probes under nonstringent conditions nd filed to hybridize with ny of the probes t high stringency. They re indicred in prentheses. MSA-I = merozoite surfce ntigen-i. x = lleles tht filed to hybridize under nonstringent conditions.? = unssigned lleles duc to mbiguous hybridiztion results. (Sequencing indicnted thzit they re colnplex mosic forrns of previously described llelic types). The thrombospondin relted nonymous protein (TRAP) typing ws done s described in the Mlerils nd Methods. Arbitrry codes (-d) Irve been given for ech series of smples nlyzed from child. NA = not vilble. 'I Vlues rc thc number of tropllozoitcsll0 leukocytes. B

10 i TYPING F SUCCESSIVE CLINICAL MALARIA ISLATES 6 1 gmetocytes crried throughout the period under study. No specific MSA-2 llele could be ssocited with these gmetocytes, probbly becuse of insufficient sensitivity of the detection. The genetic chrcteristics of the prsites crried symptomticlly before ech clinicl episode could be compred with those of the prsites collected during the episode. Smple DNA, collected during episode A, clerly differed from DNA 1. Interestingly, 0-bp MAD 20-type MSA-I llele, brely visible in the products obtined from DNA 2 s shown in Figure 5, gve strong dominnt signl in DNA, suggesting tht the prsites present t high density nd cusing episode A were present in low mounts (below the level of microscopic detection) eight dys before. This is consistent with > lo increse in prsite density observed within tht period. Smple DNA 6, collected during episode B, differed from DNA 5 collected six dys erlier, in presenting less complex picture: 60-bp KI-type MSA-1 frgment present in DNA 5 could not be detected in DNA 6, suggesting tht gin the prsites cusing episode B contined dominnt type. An dditionl pek of prsitemi (DNA 9), not febrile but ssocited with hedche, lso corresponded with the presence of prsites tht hd not been detected before, chrcterized by 290-bp MAD 20- type MSA-1 llele nd 620-bp FC27-type MSA-2 llele. In this child, the peks of prsitemi, whether febrile or not, were therefore ssocited with detection in the peripherl circultion of novel genotypes, distinct from those observed during symptomtic crrige. The detection of novel genotypes during one episode ws lso observed in the nlysis of prsites from child 22/06. The course of prsitemi in this two-yer-old child, who lso crried gmetocytes most of the time, nd typing of the prsites re shown in Figure 6. Three DNA smples (,, nd 5) were collected during the period spnning from episode A (DNAs 1 nd 2) to episode B (DNA 7). Both DNAs 1 nd 2 generted 0-bp MAD 20 MSA-1 llele nd 900- bp nd 80-bp MSA-2 lleles of the D7-type in similr proportions, suggesting tht this smple contined mixture of two prsite types presenting indistinguishble MSA- 1 lleles. Smple DNAs nd yielded identicl MSA-1 profiles nd the sme MSA-2 llele, wheres DNA 5, collected from smple contining gmetocytes nd no,microscopiclly visible trophozoites, contined single MSA- 1 llele typed s 10-bp K1-R0 hybrid. No MSA-2 llele could be mplified from this smple, probbly due to low prsite density. Smple DNA 6, collected four dys before episode B, generted the sme pttern s DNA 7, collected during episode B: 10-bp nd 90-bp K1-R0 MSA-1 hybrids. The 90-bp frgment ws more bundnt in the products mplified from DNA 7 thn in those generted from DNA 6, suggesting tht episode B ws cused by the prsites tht crried this llele. DISCUSSIN Dt described in this report s well s im other moleculr studies of prsites from Dielmolo. 1. indicte tht genetic diversity of P. flciprum prsites circulting in this villge is quite lrge. So fr, the only cses in which identicl PCR ptterns hve been observed were DNA smples collected few dys prt during the sme clinicl episode or during recrudescence resulting from incomplete prsite clernce fter tretment of the first episode, i.e., cses where the prsites re predicted to present identicl genetic chrcteristics. This indictes tht the technique used is relible both qulittively nd quntittively nd tht distinct PCR ptterns do indeed reflect intrinsic genetic differences. The PCR typing pproch used does not llow determintion of the kryotype, nd thus we do not know the ctul number of distinct clones present in the isoltes studied nor the precise gene combintion of ech of these clones. However, the results unequivoclly show tht the prsite popultions differ in the mjority of isoltes nlyzed so fr. All mjor llelic forms of the MSA-1 nd MSA-2 genes described in the literture (for review, see the report by Kemp nd others6] hve been detected in the villge. In the present study, distinct MSA-1 lleles nd 1 distinct MSA-2 lleles were observed. In cross-sectionl study of symptomtic crriers, crried out using smples collected during the 1992 trnsmission seson, 2 distinct MSA-2 lleles hve been detected.i These re miniml estimtes, since DNA sequencing is likely to detect dditionl differences. In Dielmo, clinicl mlri could be ssocited with prsite densities bove n ge-dependent threshold, determined s 275 trophozoites/100 leukocytes t one yer of ge, decresing to 191 trophozoites/l leukocytes by the ge of six yers.i6 In ll cses investigted, such levels were reched fter very rpid increse in prsite density, suggesting unrestrined prsite growth. The genetic chrcteristics of the prsite popultions collected during mlri episode differed from those detected in the blood smple collected in the preceding period. The PCR prfiles were either less complex, suggesting outgrowth of dominnt popultion or inversely more complex, indicting the presence of dditionl prsite lleles nd thus of dditionl prsite types. Whether these re dominnt is difficult to determine in complex llelic mixtures.i0 verll, however, the complexity of the infections, reflected by the number of distinct lleles detected, ws lower during the clinicl episodes thn during the phses of symptomtic crrige. The men number of MSA-2 lleles detected here in DNAs collected from clinicl episodes ws 1. per smple, while it ws in DNAs collected from children who remined symptomtic during similrly high trnsmission seson.1 This is not generl rule becuse complex popultions hve lso been observed in smples collected during mlri episode.lo The trend is, nevertheless, clerly for less complex infections during clinicl episode thn during symptomtic cmge. We interpret this s indicting tht dominnt popultion is, in fct, responsible for the observed clinicl event. Comprison of the genotypes of the prsites collected during the successive clinicl episodes experienced by child showed tht the prsite popultions collected from the peripherl circultion were different for ech episode. As summrized in Tble 2, this ws observed in three children, who experienced two episodes, in two children who hd three clinicl ttcks, nd in three children who hd four clinicl mlri episodes during this four-month period of intense trnsmission. The single cse in which the genotypes of two successive episodes could not be distinguished using the four typing rections used here ws interpreted s recrudescence. Since ech episode ws treted with quinine, +.-

11 i x i 62 CNTAMIN AND THERS TABLE 2 Comprison of the merozoite surfce ntigen-1 (MSA-I), MSA-2, nd thrombospondin relted nonymous protein (TRAP) polymerse chin rection pttems of the prsite popultion collected during successive clinicl episodes in 10 children. The specific pttems (number of bnds, size of the frgment, nd llelic type) obtined for the prsite popultion collected during successive clinicl episodes, coded A-D, hve been compred* Child MSA-I block 2 MSA-2 centrl domin TRAP cenrrl domin 02/0 A#B A#B A#B 1/05 A#B#C=D. A#B#C#D A#B#C=D 19/09 A=B#C A#B#C A#B#C 1/1 A#B#C#D A#B#C#D A#B#C#D 28/06 A#B=C A#B#C NA 02/0 A=B A#B NA 12/07 A=B A#B A#B 22/06 A#B A#B A#B 08/0 A-B A=B A=B 1/07 A#B#CZD A f B f C f D B#D * A, B, C, nd D re rbitrry codes for the successive clinicl epidodes in ech child. NA = not vilble. rpidly resulting in negtive blood?mer nd disppernce of the lleles identified during the clinicl ttck, longterm crrige of prsites ws precluded in these children. This indictes tht clinicl mlri episodes were induced by recently inoculted prsites. During the period under study, the men inocultion rte ws one infective bite every second night. With such lrge locl prsite diversity, inocultion of new strins should hve been frequent event. A longitudinl nlysis of the strins hrbored by children who remined symptomtic (nd thus untreted) during the sme period showed successive wves of geneticlly distinct prsite~. ~ This rpid turnover of strins in the peripherl circultion ws consistent with frequent inocultion of new prsite popultions. It is noteworthy tht even under such n intense trnsmission, some children remined pprently free of prsites for long periods (up to two months), presenting negtive blood smers nd no evidence of prsites by PCR (neither by direct exmintion of the PCR product nor by hybridiztion). We do not know whether this bsence of prsites indictes tht these children did not receive ny infective bite for severl weeks or whether they hd the cpcity to rpidly cler mny strins, while still being fully susceptible to other ones. The genotypes of the prsites present in the peripherl circultion during the clinicl episodes nlyzed here were ll different. This suggests tht there is no precise pthogenic type, but rther tht clinicl ttcks my be cused by lrge number of distinct strins. For some children, the encounter with P. fkiprum prsites hd different outcome for different strins. Indeed, one of the most importnt observtions ws tht in the children presenting lternting periods of symptomtic or symptomtic crrige, the genetic chrcteristics of the prsites crried t low density during symptomtic phses differed from those of the prsites cusing clinicl episode, which quickly reched high prsitemi. This indictes tht the fctors controlling prsite density in these children were strin-specific. This could reflect differences in the growth rte of vrious strins, resulting from the poor fitness of some types in certin hosts, such s, for instnce, poor invsion efficiency, impired intrerythrocytic mturtion, or slow repliction rte, ndfor reflect specific immune pressure, which would restrict prsite multipliction of certin strins while leving other strins unffected. The hypothesis tht strin-specific immune re- sponses prticipte in control of prsite density in semiimmune children is in greement with the conclusions drwn from experimentl infections in both primtesi7 nd humn~.~. This does not preclude the possibility tht mturtion of the immune response elicited by specific strin nd or exposure to dditionl strins subsequently results in protective mechnisms trnscending strin specificities. Elucidtion of the respective contribution of strin-specific nd nonspecific responses to control of prsite propgtion is key element in our understnding of protection ginst mlri. To dte, this hs been difficult to study becuse the immune effectors contributing to prsite clernce in humns re uncler, nd s consequence, the trget ntigens of protective immune mechnisms re still to be determined. Severl ntigens presenting considerble serologic diversity hve been described. The vrint ntigen locted on the surfce of the infected red blood cell elicits vrint-specific immune responses While the contribution of such n ntibody to prsite clernce remins to be demonstrted in humns, protection ws shown to be vrint-specific in Simiri monkeys. The lrge locl llelic polymorphism of the MSA-1 nd MSA-2 genes observed here nd in few other studies2i- 22 rises the interesting lterntive tht these mjor merozoite ntigensz. 2 could constitute strin-specific trgets. Investigtion of the immune response of the children studied here to vrious llelic forms of these ntigens is underwy. Acknowledgments: We thnk the Dielmo villgers who generously greed to prticipte in this study. We re indebted to L. Pereir d Silv nd to? Druilhe for essentil input in the Dielmo progrm, H. Bougnli for invluble expert ssistnce in reding prsite slides, nd M. Molyneux, V. Snewin, G. Milon, nd l? Dvid for criticlly reviewing this mnuscript. Finncil support: This work ws supported by grnts from the UNDP/World BnWH Specil Progrm for Reserch nd Trining in Tropicl Diseses nd the Ministere de l Coopertion et du Developpment. Authors ddresses: Hugues Contmin nd Thierry Fndeur, Lbortoire de Prsitologie Moleculire, Unite d Immunologie Prsitire, Institut Psteur de Guyne, BP 6010, 9706 Cyenne Cedex, French Guin. Christophe Rogier, Unite $Epidemiologie, Institut Psteur, BP 220, Dkr, Senegl. Serge Bonnefoy nd dile Mercereu-Puijlon, Unite d Immunologie Moleculire des Prsites, Institut Psteur, 25 rue du Dr. Roux, Pris, Frnce. Lssn Konte, Deprtement de Biologie Animle, Fculte des Sciences, Universite Cheikh Ant Diop, Dkr, Senegl. Jen-Frncois Trpe,

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13 FFICIAL RGAN F THE AMENCAN SCIETY F TRPICAL MEDICINE AND HYGIENE