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2002 The drug sensitivity and transmission dynamics of human malaria on Nias Island, North Sumatra, Indonesia D. J. Fryauff Malaria Program, Naval Medical Research Center, 503 Roben Grant Avenue, Silver Spring, MD
B. Leksana United States Naval Medical Research Unit No.2
S. Masbar United States Naval Medical Research Unit No.2
I. Wiady United States Naval Medical Research Unit No.2
P. Sismadi Indonesl:a11 Ministry of Health, Institute 0/ Health Research and Development
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Fryauff, D. J.; Leksana, B.; Masbar, S.; Wiady, I.; Sismadi, P.; Susanti, A. I.; Nagesha, H. S.; Eijkman Institute for Molecular Biology; Atmosoedjono, S.; Bangs, M. J.; and Baird, J. Kevin, "The drug sensitivity and transmission dynamics of human malaria on Nias Island, North Sumatra, Indonesia" (2002). Public Health Resources. 421. http://digitalcommons.unl.edu/publichealthresources/421
This Article is brought to you for free and open access by the Public Health Resources at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Public Health Resources by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors D. J. Fryauff, B. Leksana, S. Masbar, I. Wiady, P. Sismadi, A. I. Susanti, H. S. Nagesha, Eijkman Institute for Molecular Biology, S. Atmosoedjono, M. J. Bangs, and J. Kevin Baird
This article is available at DigitalCommons@University of Nebraska - Lincoln: http://digitalcommons.unl.edu/ publichealthresources/421 This article is a U.s. government work, and is not subject to copyright in the United States.
AII/lals a/Tmpic,,{ Mediciue & Parasiwlogy, Vol. 96, No.5, 447-462(2002)
The drug sensitivity and transmission dynamics of human malaria on Nias Island, North Sumatra, Indonesia
D. J. FRYAUFF*, B. LEKSANAt, S. MASBARt, I. WIADyt, P. SISMADI*, A. 1. SUSANTP', H. s. NAGESHA\ SYAFRUDDINI, S. ATMOSOED]ONOt, M. J. BANGSt and J. K. BAIRDt 'lrMalaria Program, Naval Medical Research Center, 503 Roben Grant Avenue, Silver Spring, MD 20910-7500, U.S.A. tUnited States Naval Medical Research Unit No.2, U.S. Embassy Jakana, Unit 8132, NAMRU-TWO, FPOAP 96520-8132, U.S.A. *Indonesl:a11 Ministry of Health, Institute 0/ Health Research and Development, Jalan Perceuikan No. 29, Jakarta 10560, Indonesia § Walter a'tld Eliz·a Hall Institute Jar Mecf,ical Research, fost Office, Royal Melbourne Hospital, Victoria 3050, Australia 'Eijk117an Institute Jor Moleculm" Biology, Jalan Dipo.negoro N0.69, Jakana 10430, I1'ldOltes'ia Received 19 March 2002, Revised 10 May 2002, Accepted 13 May 2002
Nias Island., off the north-western coast of Sun:iatra, Indonesia, was one of the first locations in which chloroquine resistant Plasmodium vivax malaria was reported. This re.sistance is of particular concern because its ancient megalithic cu.lturc and the outstanding surfing conditions make the island a popular tourist destination. International travel [0 and from the island could rapidly spread chloroqcine-resistanr strains of P. vivax across the planet. The threat posed by such strruns, locally and internationally, has led !O the routine and periodic re-assessment of the dli<:acy of antimalarial drugs and transmission potential on the island. Active case detection identified malaria in 124 (l7'!'~,) of no local residents whereas passiVe case detection, at the central health clinic, confirmed malaria in 77 (44%) of li3 cases of presumed 'clinical malaria' . Inforrn,ed consenting volumecl'l> who had malarial parasj.taemias were treated, accor.ding [0 the Indonesian Ministry of Health's recommendations, wfth sl11fadoxine-pyrimethamine (SP) on day 0 (for P. !alcipa",m) or with chloroquine (CQ) on days 0, I aJ;Id 2 (for, P. vivax). Each voluntj!!cr was then monitored forclinicai and parasite response until day 28. Recurrent parasitaemia by day 28 treatment was seen in 29 (8."Y,.) of the 35 P. lalcipa"wl cases ·gh'en SP (14, II and four cases showing RI, RII and RIll resis.tance, respectively). Recurrent parasitaemia was also observed, between day II and day 21, in six (21 %) of the 28 P. vivax cases given CQ. Although the results of quantitative analysis confirmed only low pTi;!valences of CQ-resistant P. vivax malaria, me prevalence of SP resistance among the P. Ia/Cipa171111 cases v.>as among the highest seen in Indonesia. When the parasites present in the volunteers with P. /alcip(mml infections were genotyped, mut;ltiQ.ns associated with pyrimethamine resistance were found at high frequency in the dhJr gene but there was no evidence· of selection for sulfadoxine resistance in the d/zps .gene. Night-biting mosquitoes were surveyed by human landing collections and tested for sporozoite infection. Among the five species of human-biting anophclines collected, A.llopheles .~lIl1daiCII:< was dominant (68%) and thl: only species found to be infective - two (1 .2%) ef ) 67 females being found carrying P. vi~'ax sporozoites. The risk of malarial infection for humans on Nias was considered high because of the abundance of asymptomatic carriers, the reduced effectiveness of the available antimalarial drugs, and the biting and infection 'rates' of the local All. sulldaiClls.
Malaria caused by Plasmodium vivax occurs an immense burden of human suffering and over a broader geographical range than that illness. Chloroquine (CQ) has long been a caused by P. Jalcipanml and accounts for safe, inexpensive and reliable treatment for P. vivax malaria but there are accumulating Reprint requests to: D. J. Fryauff. reports of treatment failure from such diverse E-mail: fryaufJ'd':<:{! nmrc.navy.mil; fax: + I 3.0 I 3197545. locations as Papua New Guinea (Rieckmann " 2002 The Li v,c:rpool School ofTropicnl Medicine 001: 10.1179/000349802125001249 44 8 FRYAUFF ETAL.
('I al., 1989), Indonesia (Baird et al., 199 1b; MATERIALS AND M ETHODS Schwartz el al., 1991 ), India (Garg el al., 1995), and Guyan a (Craig and Kain, 1996). Study S ites a n d Subjects International travel, which has becom e mu ch All-age malaria screenings we re co n easier and m ore frequent over the last du cted in five rural co mmunities (Lagundri, four decades, may figure more importantly Walondrawa, Botohilitan o, Hilinifaoso and in facilitating the geographi cal spread of Hiliamaetaniha) aro und the port of T eluk CQ-resistant P. ,·jvax (CQ RPV) than it D alarn, on the sou thern coast of N ias Island , did in spreading CQ- rcsistant P. [alcipannn between the Ju ly and September of 1998. (CQRP F) in the 1960s. Ni as covers an area of 56 25 km". D etailed , confirma tive stu dies of CQRPV Passive case dete ction was also performed, have been relatively few, and efforts to track on a da ily basis for 3 weeks (1-2 1 August the geographical spread of thi s pr obl em 1998 ), through the health clin ic at Lagundri, have been largel y co nfined to the Indonesian which covers all five study co mmunities. archipe lago - a popul ou s nation of some M ost of the residents in the area have 140,000 islands. Am ong all of th ese islands, occupations that are agricultural (rice culti Ni as, in north- western Sum atra, drew early vation, patchouli cultivation and perfume-o il attenti on as one of the first areas on the processing ) or associated with tourism . The planet th ought to be affec ted by C QRPV. popularity of Legundri as an international As Nias draws large numb ers of interna tional touristfsurfing des tination has created an tourists to its antiquities and surfers to its increasing demand for lodgin g, food and waves, was apparent that there was a great it othe r services. T ourist lodgings, restaurants risk of resistant strains of malarial parasite and housing for Indonesian families and on the island bein g carried all over the globe . workers have proliferated on the land between Following on the case report of CQRPV in the rice paddies, mangrove fore st and a repatriated Am erican tourist who acquired beach, with faciliti es ranging from extremely his infection on Nias (Schwartz et al., 1991 ) inexpensive/basic to we stern -style, 'five-star' and confirmation of th is CQ RPV at the luxury. 1\ 1any young tourists appear to Centers for Disease Control and Preventi on , opt for very simple and affo rdable, longer in Atlanta (Collins et al., 1992), in-vivo field term lod ging in traditional-style bungalow s. testing of CQ sensitivity/resista nce was co n Mosquito coi ls and untreated bednets are ducted in early 1995 (Baird er al., 1996). widely used by both local resident s and tour The results of thi s first survey revealed CQ resistance, to suppressive levels, in 33% of ists. CQ, SP and quinin e are the respective the P. oiuax investigated, with parasitaemi as first-, second- and third-line drugs for malaria recurring, within 19 days of supervised treatment in the Indon esian N ational Health therapy with CQ of good qu ality, in 14 % Service and all are locally available in the (Baird el al., 1996). The ma in aim of the shops . present study, conducted 3 years later at the same site, was to re-assess the sensitivity of local P. viwax to CQ ill vivo, to det ermine Sc reening and Enrolment whe ther the frequ en cy and degree of CQ Indi viduals with un complicat ed , slide resistance had measurably increased since the confirme d malaria who we re aged > 4 years, first survey . At the same time, the efficacy, abl e to swa llow pills, not pregnant and who in vivo, ofsulfadoxine- pyriruethamine (SP) had not consumed antimalarial drug s or the seco nd- line therapy for uncomplicated antibiotics in the previou s week were invited P. [alciparunt infection - and the risk of to vo lunteer for malaria treatm ent in the malarial infection faced by a joint Am erican co ntext of a 28-day, in-vivo test. Written, Indonesian military exercise planned for the inform ed co nsent \vas ob taine d from eac h area were evaluated. volunteer or the volunteer's paren t/guardian . .\ !AU RIA ON KIAS ISUND, S U~IATRA 449
This consent included an assurance th at th e recorded and fingerprick blood sam ples (for volunteer agreed (1) to use no ot he r drug or thi ck and thin blood smears) were collected treatment but those provided and supervised on days 0- 4, 7, II, 14, 18, 21 and 28 and by th e research ers, (2) to submit to daily also at any time of illness indi cative ofmalaria. sym ptom evalua tions and fingcrp ricks, and The blood smcars were stained with Giemsa (3) to submit to venipunc ture for cases of and examined by oil-immersion , light micro P. uiuax that reappeared within 2 1 days of scopy (at x 1000 ) by a skilled microscopi st. receiving CQ . An enrolment of 38 1'. uiuax Each thi ck smear was examined for malarial cases was sought, based upon th e proportion parasites and only considered negati ve if no of CQRPV previously reported ( 14% ;Baird parasites were seen in 300 fields. Sexual er al., 1996), and th e aim to estimate the and asex ua l-stage parasites were separately new proporti on, with 90% confidence , to counted against 200 leucocytes and each be within 10 percen tage points of the true count was then multiplied by 40 to give value (Lcrneshowand T aber, 1991).With no an estimate of the number of pa rasitcs/ul previous measu re of clinical or therapeut ic blo od (assuming eac h subject had 8000 resistance to 51' in the local P. fakiparuni, it leucocytcs/ul ). was arbitrarily assumed that treatment failure Aliq uots (I 00 ul) of fingerprick blood would occur in 25% ofcases (the critica l level ob ta ine d on day 0, 2 and any time of above whi ch an alternate treatment drug suspected treatment failure were collected wo uld be advised). With 90 % confidenc e onto filter pap ers, labelled and air dri ed for and 10(% precision , th e desired samp le size subseq ue nt use in ana lysing blood conce n for testing the sensitivity of local P. falcipa rtnn trations of CQ or th e parasite geno type to SP was the n estima ted to be 5 1 infecti ons (sec below). Unremitti ng pa rasitae rnia or (Lemeshow and T ab er, 199 1). Individuals reapp earan ce of asexua l parasites during the with parasitaernia who de clined to enrol or 28-day pe riod of the in-v ivo test consti tuted who were excluded from enrolme nt were a treat ment failure. The clinical symptoms provided with free treatment and follow-up, of malaria, with parasi taernia tha t did not according to the local standa rd of care. resolve following treatme nt or which recurred with parasitaernia, during the 28-day. follow up period, constituted a clinical failure. In C h e m other a py a n d FoUow-up acc ordance with nati onal health policy in Each subjec t with P. cioax infection was Indonesia, any CQ and SP failures observed given a total of 25 mg CQ base (ResochinT.,,; were given ora l qui nine for 7 days, as rescue PT Bayer, Jakarta, Indonesiaj/kg body weight, treatment . Cumulative indices of therapeu tic as three oral doses of 10, 10 and 5 rug/kg ( parasitological) and clinical failure for ea ch 0, 24 and 48 h afte r rec ruitme nt (i.c, days Plasmodium species and dru g trea tment were 0, I and 2), resp ectively. The subjects calculated using the life-table method . The with P. [alcipannn infection were eac h given parasitological responses of P. [alc iparum to a single ora l dose of SP (Fansidar " ; Roch e, SP wen: gra ded, according to the standard Basel, Switzerland) - just on e tablet classification ofthe World Health O rgan ization (cont aining 500 mg sulfadoxine and 25 mg (1990), as S, RI , RlI or RIl l. pyrimethamine) if they weighed 15- 19.9 kg, and 1.5, 2.0, 2.5 and 3.0 tablets if they weighed 20-29, 30 - 39, 40- 49 and > 50 kg, .I\ l ea su r cln cn t of Blood C onccn tr a ti ons respectively. All the tabl ets were cons umed of CQ after a sma ll snack of sweet biscuits and CQ and its principal metabolite, mono water and each treatment was witnessed desethylchloroquine (D CQ) , were extracted, by a member of the study team. Ph ysical from the filter-paper blots of the whole complaint s and axillary temperature \'vere blood samples of those tre ated with CQ , so 45 0 FRYA UFF ET AL. that their con centration s co uld be me asured nuclease digestion, purified lambda DNA by HPLC (Patchen er al., 1983; Fryauff (Prornega) was used in eac h RFLP exp eri et al., 1998). T he persistence or reappear ment. D igested produ cts were subjected ance of asexual-stag e P. rioax parasites, to electrop horesis on 1.5%- 3%, aga rose gel, following full, supervised treatment and in or ultrapure agarosc gel (Progcn, Darra, the presence of whole-blood co ncentrations Australia) for enhanced resolution . The of CQ + O CQ that exceeded 100 ng/m l PCR products of interest were sequenced the estimated minimal concentration effective according to published method ology (Wang against CQ-se nsitive strains of this parasite et al., 1997a, b), to verify the RFLP results (Baird ct al., 1997) - was taken as con and to identify any new mutation sites in the firmation of CQ resistan ce. [N ot e th at rhis dhfr and dhps products. definition may apply to post-treatment para sitae rnias arising from recrudescence, relapse of Transmission or re-infection (Baird et al., 1997).} Assessm en t by Mosquitoes M osquitoes were captured outdoors in each of the five study communities, between Molecular J\\arkers of SP Resistance in 18.00 and 23 .00 hours, as they landed on Plasmodiurn falciparurn the memb ers of a two-man team equipped Parasite D NA was extracted, from the filter with flashlights and aspirators. Anophelines pap er blots of the whole-b lood samples of were sorted, identified to species and stored those treated wi th SP, usin g 5%) Chelex- IOO dried over silica gel until their heads and resin (Bio-Ra d Laboratories, Hercules, CA) thoraces could be tes ted in an ELIS A for in distilled water and then purified through Plasmodium sporozoites (\Xlirtz et al., 1985 , an A72 1C minicolumn (Prornega, M ad ison 1992). 1\l onoclonal antibodies for Pifalcipan un WI). T he DNA was then amplified using the and the circumsporozo ite variants of P. vivax nested-Pf.R method described by N agesh a (VK24 7 and PV2 1O) wer e em ploye d in the er al. (200 1), the appropriate po sitive and ELIS A. The ento mo logical data were used neg ative controls (to minimize the risk of to produce estimates of the man-biting rate contamination error in the first and second (bites/person-night) and the entomological rounds of the peR), and conventional inoculation rate (infective bites/person-month) primers and those speci fic for the resistan ce assuming that the local mo squit oes only fed associated mutation s in the parasite's dhfr durin g the collect ion period (18.00- 23.00 or dhps genes (O urasingh et at., 1998). The ho urs ) and that all the mosquitoes tha t dhfr and dhps polymorphisms were ident i landed on the hum an bait wou ld have fed fied by RFLI' (restriction-fragment-length on the bait. polymorphism ) ana lysis. The nest ed am pli ficat ion products of dhfr were digested with NlaIIl, Tall, Tsp509 I, X 1//1/ 1 and [)l'al , RES U LTS for discriminating the alleles at cod on s 16, 50, 5 1 and 164, whereas codo n 108 was Malarra Prevalence by Location and selectively cleaved with AIII I, RSI'I and SerF!. Age-group The dhps PC R produc ts were digested with All-age active case detec tion (ACO), iV/spA ll, Avail and Fok l fo r the identi conducted in July- Augu st 1998, identified fication of mutation s at codo ns 436,437 and malarial par asitaern ia in 122 ( 17.2%) of the 540 ,B. T ABLE I . The results of malaria p..1im-prt·i:ollclla screening by <1((i"", e.lSt' detection lUI 111.' unuhcru cwst of X i<1s Island, Su matra. 1I1JOII.'s;d, ;/1 JI/~\L.. S.·p t .: m b t· r / 998 >-:0. and (%) of subiects: round positive for; Malarial Pfasmo,JiwlI P/ Hilinifuoso 258 2-1 «() 7 (3) 17 (7) o (0) Lagundri 1-13 9 (0) 0 (0) 9 (0) 0 (0) \'·a!ondrawa 1 0~ 26 (25) i 3 (13 ) 13 ( 13) 0 (0) Botohilitano 76 i 2 ( i O) Q ( 12) 3 ( ~) 0 (0) Hiliam acranih a 12Q s: (·10) 29 (2 2) 20 (16) :2 (2) All rive study sites 7 10 122 ( 17.2) ')8 (R.2) b2 (8. i ) 2 (0. 1) 27 (8.6%) of the 314 appa rent ly asymp likely to be confirme d positive by micro tomatic schoolchildren (aged 6- 13 years) scopy as those who were younge r or olde r) screened; six (1.9%) appeared to have pure with a relative risk (RR) of 2.14 and corres P. fa lciparunt infections whereas th e other ponding 95% confidence interva l (CI) of 2 1 (6.7%) appeared to have pure P. ".'h'ax 1.30 - 3.54. infections. Passive case detection (PC D) , over 3 weeks in the Lagundri hea lth clinic, con finned malaria in 77 (44.8%) of the 173 cases E ffica cy of the CQ Treatment of judged to be of 'clinica l malaria' by local Ptasrnodiurn vivax Infecti on healthcarc provid ers. The confirm ed cases Illness at enro lment - most commo nly hea d appeared to be infected with P. [alcipan nn ache (76%), fever (68%), malaise (44%), only (47 case s), with 1'. vicax only (28 cases) or a mixture ofth ese two species (two cases). nausea (·10%) an d muscle pain (24%) was reported by 26 of the 36 subjects with For both P. 'i..'i'iJa x and P. falciparum. th e age distributions of those found pos itive P. virax infection and eight were found to by ACD differed markedly for those found be febrile (i.e. with an axillary temperature positive by PCD (Fig. I). Children aged < 6 ~ 3 7 . 5 C). The cha racteristics of cases who years, for example, accounted for 23'Yo of recei ved CQ treatment and remained avail the infections found by ACD screening but able for all or most of follow-up peri od are made up only SIX, of the confirmed clinical presented in Table 2. The corresponding malaria cases in the PCD (1'= 0.007) . therapeutic and clinical responses are sum Despite the high freq uency of infection seen marized in Figure 2. Baseline (pre-treatment) in the children screened during the ACD concentrations of CQ + DCQ in three of and the predominance of children in the th e 30 subjects tested ranged from I 13 demography of the population (77% of the 639 ng/rnl, ind icating recent previou s treat residents of the study communities were ment and/or potentially resistant infec tions. aged < 16 years at the tim e of enrolment), All parasitaernias declined rapidl y in response adults (aged > 15 years ) made up 52 tX, of to th e CQ trea tment given as part of thc the d iagn osed cases of clinica l malaria in the present study and cleared by 96 h. The mean ACD and accounted for 43% of those CO l1 parasite-clearan ce time was 36.7 h, with CI finned by mic roscopy in the PCD. In the of ± 5.6 h. Illness aba ted in the majority of PCD, the olde r children and young adults the subjec ts by 72 h, with only three of 35 (i.e. those aged 6- 40 years) were twice as still reponing head ach e or nau sea at that 452 FRYAUFF ET AI.. SO 70 (u) (J 60 - ~ (J ==:: 50 ;!; 40 ~ 30 .0-. &: 20 10 0 0-5 6- 10 11-15 16-20 2 1-40 >40 (N=22) (N=34) (N=27) (N=23) (N=36) (N=3 1) Age-group (years) SO 70 (b) u 60 ~ ..u= 50 ev: 40 =:: 30 0- ~ 20 10 . o. 0-5 6-10 11 -15 16-20 21 -40 >40 (N=SO) (N=265) (N=131) (N= I S) (N=83) (N=43 ) Age-group (years) FI(' . 1. The proportions of each age-group, screened by passive (a) an d actin: (0) C3<;C detection. found to be infected with PI..umodium ! lime . The first reappearance ofasexual para had 160 trophozoitcs/ul on day II Cr able 2) sites following therapy and clearance was and CQ + D CQ concentrations uf 0 and on day 11. in two asymptomatic children. 270 ng/ml, respectively, on day 12. The only These 1WO children, who had had 7280 and ot her recurrent purasitaeruias detec ted, on > 40 ,000 tr ophozoites/ul and correspon d day 18 in another four subjects. wen: all found ing CQ + D CQ concentra tions uf 23 5 and when those affected had low or undetec table 57 ng/ml on day 0 ( pre-treatment), both conce ntrations ofCQ + D CQ in their blood . .\ tALA RIA ON NIAS ISLAND, SUMATRA 453 TABLE 2. SIIIIII1IJ ry of I1h' results of the 28-JolY. in- rico tests of the sensitivities of Plasmodi um falciparum ro SIlIf"Jox ;Il~' -pyn'mt'lh l1 m ille (SP) .m J P. vivax to d ,/onJi/lI;lle ( e Q) Characteristic x e . of subic cts 39 36 No. of mal es : femal es 22:17 22 : 1-1 1>AV-o ( I · M E -T REAT.\ I E ~"'T) VALL'""ES ;\t t'an age, (m edian) and (range] ( years) 15.6 (1 2) [6- 55 ] 1-1 ( I I) 1-1 - 501 ~t c a n weight. (m edi an) and [range) (kg) 33.8 (35) [15 -571 29. 3 (27. 5) (12-19) .\tean haemoglobin conc entration and 9.7 (9 .2- 12.2) 9.5 (9. 2-Q.R) (q')% confidence interva l)(g'dl) Geometric mean parasitaemia and (ra nge) 100 3 (40-80, 000) -18 3 (-10- -10, 000) (a..exual parasites'ul ) CU ~ l nJr. T I \· E I~ CID E SC E OF PAR.",SITAE.\lIA Day 0 0 .00 0 .00 Day" 0 .23 0 .00 Day 7 0 .34 0.00 Day 11 0 .59 0.06 Day I-I 0 .69 0.20 Day 18 0.69 0.20 Day 21 0.73 0.20 Day ~8 0.76 0.20 Me an time to recurrence and (range) (days) 11.0 (·1-28) 17.0 (12 - 19) MEAI' CQ + DCQ CONC E:-ITRATIONS ANn/OR ( RAKGE) (ng/m l) Day 0 - (0- 633)" Day 2 568 (2 16- 1169) Day 2R 38 (0-3 15)' Day of recurrence -(0-270)* * Blood samples collected from 30 of the subjects at enrolme nt revealed that seven of the 30 then had detectable blood concentrations (22- 633 ng-ml ) ofCQ plus its metabolite rnonod cscth ylchloroquine (D CQl , and that three of th e seven had > 100 ng CQ + DCQ'ml. "Seven of th e 22 subjects chec ked had detectable blood concentrations of CQ +- D CQ on day 28, three having > 100 ngml at that rime . t O ne of the six subjects checke d on th e- day of recurrence had > 100 ng CQ + DCQ'm l. Efficacy of the SP Treatment of SP were still complaining of headache and Plasrnodiurn folciporum In fection malaise, respectively. Asexual pa rasitaernias Symptom s - most commonly headache declined by > 25 % within 48 h of treatment (94%), fever (8 1%), malaise (72%), nausea in 34 cases (of the 38 providing the relevant (56%), and vomiting (37%) - were reported data) and had clea red by day 4 (96 h) in at enrolment by 33 of th e 39 subjects with 28 of the 39 cases. U nremitting R1II- or P. falcipa nnn infection although only 1-1 Rll-typ e responses within 7 days of treat (36%) were found febrile at that time. The ment characteri zed, respectively, four (I 1%) geometric mean (G :\ I) P. falcipannn pa ra and I I (29%) of the SP treatments. Two of sitacmia before treatment (on day 0) was 10 the subjects treated with SP withdrew from times higher [han the corresponding P. riocx follow-up and three others were dis-enrolled parasitaernia (T able 2). In general, the between days 7 and 11 because ofintercurrent clinical and therapeut ic resp onses to SP were P. V iVdX infection. Figu re 2 illustrat es the poor. At the day-3 (72 h) follow-up, fever cum ulative incidence of clinical and the ra had resolved in 13 of the 14, initially febrile peut ic failures over th e 28 days following cases but 47 % an d 24 % of all those given S P trea tment. T he majority (24/29) of the 454 FRYAUFF ET AL. 100 90 so . --' .- 70 40 30 .d 20 10 o . ... . - -0------0------0 o 7 I I 14 IS 2 1 28 Duy FIG . 2. Profiles of the clinical and parasitological responses obs erved in the sub jects post-treatment."I1H.' lines plotted show the proportions ofsubje cts who wen.' symptom atic (L) or parasitacmic ( _) after their P/tJ sl1todill 1J/ !l1ld p,mIlJl infections were [rcated with sulfudoxine-pvrimcthammc (on da y 0) and the corresponding pr oportions of subjects wh o wer e sym pto matic ( . ) or pamsitaemic () aft er th eir P. vinax infections were treated with chloro q uin e. therapeutic failures occ urred during the first or patte rn of these dhf r mutation s and th e 11 days, with treatment success achieved in in-vivo resp on se Cr able 3); the double muta only six of the 35 ease s wh o provided suffi tion, for example, was seen in each of th ree cient data . The frequency of gam etocyte sensitive (S) infecti on s as well as in th ree carriage and th e G l\1 densit y of th e gamcto RIll treatment failures. The sing le infecti on cytaernias observed inc reased progressively that was wild-type at all dlifr codo ns appea red over th e first 7 da ys following SP trea tment to recrudesce on day 11 post-treatment. A (Fig. 3). Retrospective mole cular ana lyses, comparison of the day-O and day-7 dhfr to determine wh ether allelic mutations in th e profiles of each of nine cases demon strating dhps and dhfr genes of the infecting parasites RIl- or RIll-type resistan ce showed the could have predi cted SP treatment outcome, Arg59 and Asn I08 double mutati on at both were performed on 27 cases wh o had be en tim e-poin ts. Quinine rescu e ther apy was graded S, RI, RII or RIll. Sequencing of the successful for all of the SP treatment failu res. peR products revealed that dhps codo ns 43 6, 437, 581, and 6 13 were all normal wild -type and that mu tati on s in th is gene had not arisen or been selected for. Although Malaria Transmission Potential sequencing ofdhfr identified point m utation s H uman-biting A nopheles accounted for 25 1 only at codons 108 and 59 , th ese m utations ( 12.5%) ofthe 2003 night-b iting mosquitoes were dete cted in 26 (96%) and 2 1 (78%)) of captured during 2 16 person-hours of collec the 27 cases, respectively, with 20 cases eac h tion. Five ano pheline species, including three having the double mutati on . There was no kno wn to be vectors of P. fal ciparum and/or apparent asso ciation between the frequen cy P. uiuax - A nopheles sundaicus (Rodcnwalt) , MAL\RIA 0/\ NIAS ISL,,""O, SU,\ lAT RA 45 5 100 500 ~ '<, 450 'f. OJ 80 400 s, :Jc :J - 350 OJ ~ ::: ~ .. ~ 60 - 300 -~o -c-, .... '-c :J 17 ,..; c -- 250 ~ :J r-~ c ~ ~ 40 200 - Cl; ./ U ~ ~ ~ ~ ~ 150 :J "- ri 20 100 ::: c 50 :J CJ () , 0 o 2 3 4 7 Days post-treatment FIG . 3. Effect of sulfado xinc- pyr imerharnine treatment of l'/"Hlllodiu1I/ fuldpunmt infection s un [he prevalence of (detectable) gametocytaemia ( - ) and the geome tric mean densities of P.[alcipartnn garnetocytcs ( . ) amo ng [he garnctocytacmics. A n. sinensis (W iederuann) an d All. tessellatus DISC USSION (T heob ald) - we re ide ntified. Anopheles sundaicus accounted for 68 % of the 245 The principal aim of the prese nt study was Anopheles identified and tested and An. sinensis to initiate periodic, in-vivo re-testing, to de tect for 18%. Collectively, Anopheles biting rates any cha nge in the freque ncy of C QRl'V at ranged from I - 20/person-night, with All. an Indonesian site where th is trait had been sundaicuspredominant in 11 ofthe 18 location s first recogni zed and co nfirmed (Schwartz surveyed and All. sinensis predominant in six er al., 1991 ; Collins <'I aI., 1992 ). The pre inland locations. sent investigation, cond uct ed 3 years after Only [\\'0 mosquitoes - both All. sundaicus the first survey for C Q RPV on N ias (Baird infected with the Pv210 variant ofP. "im x et al., )996), does not provide any evidence were found to be carryi ng spo rozoi tes. The of such a change . Althou gh the present sporozoite 'rate" was therefore 0.8% for results inclu de clear indication s of P. 'V;V QX all of the Anopheles tested and 1.2% for resista nce to both suppressive and thera th e A ll. sundaicus. Since the mean biting peu tic co ncentra tions of CQ, they show no rate for all Anopheles was 6.8 bit es/person grea ter freq uency of such resistance than that night, the entomo logical inoculation rate was reported for the same site in 1995 (Baird et al., (conservatively) estima ted at 1.6 infective 1996). The C Q-lOlerant or C Q-resist ant bites/person -m onth (T able ~ ) . P. cicax troph ozoitcs observed in the present 456 FRYAUF F ET AI-. TABLE 3. 17,. wild-typo' alleles ( )) ami allelic mlfhuiollS ( e) .!aCt"',} ill ,lit' K.'/lCX for dihyd"ofo!,l/o,' l'.:dlh'/I1W (pfdhfr) and tliltydropreroarc syutlcctvse (Pfdhps) ill Pla smodium falciparum i:w!ul.'S /tmll the subjects (r.'il l.'ti n';11i J 1/((<1dtJX ill ,'~!'J"';1IIi!!11 ,111I 111 " Pjdhp., co don PNhj r cnu ull Patien t no. Clinical respon se -l37 'lSI h l1 I " "59 HlH I O~ PUt 2R RI (d ay 1-\.) PLG --I 7 RI (d ay I~ ) •• PLG 51 RI (d ay I I) •• PLG 63 RI (d ay II) •• , PLG !Ol) RIII •• PL G 139 RI (day 2 1) --, ; r:•• PLG 19 3 RI (d ay II) ,; ~ PLG 199 RlI ; -, •• 2 15·) RI (d ay I I) - •• 2 1'56 S r •• 2 16 1 RI (d ay II) , •• -, 30 50 S r • • 3057 S • • -, ,•• ·100·1 RI (day I t) - -100 6 RI (day I I) PL G \2 ·1" RIll ( PLG \·l l · RII • •-, I'I. G 197· RII Pl.G \ 9Rt RIII • • 6007* RII r •• QU7 1· RII •• 500 3· RII •• b05 1" lUI •• 600·1· RII •• 6060· lUI 20Y' lUI • • 607 1'" lUI •• ·111 ~· cod ons of isolates collected from this pa tient on da y (1 and the da y of recurren ce were compared (and found to be ident ical). 'J'ABLE :L 'J 'h ~' pveoatcnces lIt" ill/ ~' a i oll >:1..'1[il ma larial sporozoite, man-biting r!,lUS (.\IB R) , ami estimated cutomotoeical inoculation rates ( EIR) for f h ~' Anopheles III 11S41';f/)~'S collected 011 .\ 'ius /slalld, S umatra Sporozoite ELISA ~IRR ' EIR' Spec ies No. rested No. an d ('Yo) po sitive (bites-person -n ight) (infective bircs-person-mourh) .-111l1phdes f<'ss.:lla flut (, 0 (il) 0.2 0 / 111 . kllchi I J 0 (il ) 0." 0 All. mmfvrdi I ·l 0 (0) 0..1 0 A'l . s i ll o l.\' i~t "; 0 (0) 1.2 0 All. wllduiclISt 167 2t ( 1.2) -1.6 1.6 ..Assuming all bit es occur each night on ly during the 'i-h peri od ( 18.00- 2 3.00 hours) when the landing catches were made and that 311 landing rno squirocs would ha w bit ten th e human bait . For [he .\l HR, a month was taken to be 28 da ys, t C onfim1cd vector (If at least \1Ot: I Y.,WIIOJill ll/ species tha t infects h umans . t Both Infected with PillS/lIlll-filllll »ico». MALARIA 01'\ :-lIAS ISL-\l'D, SU.\ L-\T RA 45 7 study were also found in the pre sence of criterion for a cha nge in the an tima larial CQ + D CQ conc entrations similar to those drug used (WHO , 1994 ). All 74 cases fro m detected in 1995 (Baird 01 al. 1996). central and nonhero Sumatra who were It is unclear wh y, given the apparently tested i ll V itl{l between 1981 and 1985 equ al selective pressure applied world-wid e dem onstrated sensitivity to SP , although to P. viuax and P. [alcipurnnrby CQ use and in-vitro testing over the same period and in mis-u se, CQRPV is much less common than the same locations sho wed SP resistance in CQ RPF. This may be related to the obse r 28 of 38 isolates (T iitra 01 al., 1997). A rapid vation that CQ-scnsitive P. oivax can be killed ch ange in the sensitivity of P. [alcipa rum by a dose of CQ that is only abo ut a qu arter to pyrimethamine and SP was obse rved in of th e dose needed to kill CQ-sensi tive north- eastern Papuan Indonesia (H offman P. [alciparum (Berliner 01 aI., 194 8). The CQ 01 aI., 1985 , 1987; Baird 01 al., 199I a), resistan ce in P. falciparum has been shown altho ugh resistance to pyrime tha mine and to be cent rally depe ndent up on a point cross-resistance to proguanil evolved early mutation (K76n in a single gene (pfm ) and progressed rapidly there, as a res ult of the coding for a transpo rt protein in membrane mass dist ribution of pyri me rha mi nizcd salt of the parasite's digestive vacuo le (Fidock in 1959-1 960 (M cuwissen, 1961 ). Interest (1 al., 2000). This point mutati on was pre ingly, th e ca mpa ign to distribute and pro sent in every clinical case of CQRPF studied mote the use of this med icat ed salt mark edly in M ali by Djimde 01 al. (200 1) and in 93% reduced the prevalen ce of P. vivax and of the CQ RPF cases studied in Papua, P. nratariac malaria, with pyrimethamine Indonesia, by Maguire 01 al. (200 I). Alth ough remaining effective as both a treatment a gen e ortho log present in P. uiuax ( p'vcg I 0) and prophylactic against P. cioax at a time is remarkably similar to pfcn in terms of its of increasing resistance in P. [alcipan tm position , com position and struc ture, no point (Mcuwisscn, 1964 ). There appears to be no mutati ons th at distinguish CQ-sen sitivc and record of the use of pyrirnetharninizcd salt CQ -rcsiswnt ph enotypes of P. ciua» have on Xias. Resistan ce to CQ in P. faiciparum been found in pucgl O(N omura Cl al., 2001 ). and high freq uenc ies of gametocyte carria ge The apparent molecular divergence in the in chi ldren pr ompted health ollicials and C Q-resistance strategies of P. falciparum and researchers to ado pt SP plus primaquine for P. viuax is in contrast to the commo n mech the routine treatment of malaria in coastal anism of antifolate resistan ce seen in all th e north Sumatra during a community health mal ari al parasites of rodents and humans project in 1985 -1989. Alth ou gh this co investigated (D e Pecoulas ct al., 1998; operative (Indonesian-Japanese) programme Carlto n 01 al., 200 1). led to a marked reduction in the prevalen ce The present results do not warrant of P. falcipanun infection, there was littl e increased concern about CQRPV on Nia s change in P. oioax, despit e the combine d usc or more frequent monitoring of CQRPV on of chemo the rapy, bednets and larvi ciding the island. H owever , the poor clin ical and (Doi 01 al., 1989). therapeutic effects ofSP on local Pi fa lcipannn The results of th e present analyses of dhfr and the almost ubiquitous occurrence of dhfr and dhps in P. falciparum from Nia s are mutat ions conferring pyrimethamine resist singular in dem onstrating no evide nce of any ance in these parasites arc worrying. The mutati on in the dhps gene associated with frequency of SP treatment failure obse rved SP resistance. The non-involvemen t of dhps on N ias is among the highe st reco rded in point mutations appears not to have been Indonesia - even excee ding those recorded reported before in any area were the re were on Papuan Indonesia (Baird et al., 1991a; such high freq uencies of SP treatment failure. Tjitra 01 al., 1997; N agesh a 01 al., 2001) The dhps mutat ion at position 437 is a com and meets the World H ealth Organ izat ion's monly reported marker of sulfadoxine pressure 45 8 FRYAUF F ET AI.. and ' v·as detected in all the P. fulcipannn higher. In compariso n, on an isolated island isolates, from nearby M alaysia, tested by Cox off north- western Papuan Indon esia, whe re Sin gh et al. (200 I). Analysis of dhfr from all the P. [alcipanun appeared Sl'-sen sitive, P. falcip arum collec ted in Papuan Indonesia garnetocy tes wer e see n in only four of 40 ide ntified the A SH 108 mutation in 8 1% of cases between days 0 and 7 and in only 12 of malaria-naive immigrants (N agesha et al., 288 smea rs prepared later in the follow-up, 20 0 I) and 62%, of malar ia-immune na tive and then always at low densities (Fryauff et al., Papuan s (H . S. Na gesha, unpubl, obs .); 1999). No atte m pt was made to determ ine the both significantly lower proportions tha n the viability and infectivit y of the game tocytes frequen cy of Asn I08 in the present samples in the elevated gamctocy tae rn ias appa rently from N ias (96%).In contrast to th e pre induced by SP treatmen t on Ni as, but these sent results frum Ni as, the P. [alciparnni in gametocyt es may well be viabl e and similarly the sym pto matic, non-immune immigrants resist ant to the spo ronticidal activity of 5P from Papuan Indon esia com monly expressed (Butcher, 1997 ; Robert et al., 2000). T he point mutation s in the dlips gene at codons early appearance of the garnc to cytacrnias, 4 37 (83%) and 54 0 (47%), altho ug h " lips soon after SP treatment , has been taken as mutation s (at position 437 in 24 %, an d at an ind ication tha t the gametocytcs seen, many pos itio n 54 0 in 5%) were seen far less of which arc immature and non-infective, frequently in th e parasites from th e asym p ha ve not been induced by the treatmen t but to matic infections in the native Papuans merely mobilized or re-di stributed from sites (H . S. Nagesha, unpub!. obs.). It see ms whe re they have been seques tered (T argett possible that dhps mutation s pla y a relatively et al., 2001). Whil e the pyrimethamine small role in SP resist an ce (or confer a me di ated mobilization or development of relatively sma ll adva ntage in terms of the garnetocytc s (C hurmongkonkul et al. 1992) pa ras ite's survival) under conditions of low on Ni as may h ave been enhanced by muta transmi ssion int en sity. tions in the d/~fr gene , it also see ms possible A recently reconside red problem is the that the absence of mutations assoc iated potential for 5P treatm ent to exacerbate with sulfadoxinc resistance in dhps m ay co m m un ity levels of P. [alcipartnn tra ns have somehow stim ulated gamctocytogenes is mi ssion, by elevating the prevalence and (Butcher, 1997; Hogh el al., 1998). inten sity of ga rnctocyta cmias. This d is T he present dem onstration of P. uioax advantage of SP ha s alread y been reported sporozoites in All. sundaicns and th e domi in Asia (Tin and Nyunt Hlaing, 1984; nance of this mosquito species among the Marworo "I al., 1986) and much debated man -bi tin g ano phelincs collecte d were not (Ittiravivongs et al., 1984 ; Strickland et al., unexpected in the coastal study area. Anopheles 1986; Baird el al., 199I e). Among the non sundaicus is conside red to be the dominant irnrnuncs in north- eastern Papuan Ind on esia vec to r of both P. oioax and P. [alcipanun who were given SP in 1996 -1 998, as a over m uch of coas tal Indon esia (Imai t!t al., resc ue treatmen t following CQ failure, the 1988) and is respon sible for the persisten ce prevalenc e of garnetocytaernia rose fro m 0% of malari a along the sout hern coast of Java to 43 % within 7 da ys, with the GJ\\ int en sity despite the successful eliminat ion of malaria (among th ose who were gamerocytae mic ) from most of this island (Atm oesodiono et al., peaking, on day 14, at 199 gam etocytes/ul 1992). A nopheles tesselatus is a confirmed vector (C I = I 53- 277 lfLi ; unpub!. obs. ). H owever, of P. on-ax and P. fukipannn in south ern Asia the gametocyte resp on se obse rved in N ics, (G arnage-M end is et al. , 1993) and was pre where the prevalence ofgam ctocytaem ia rose viously the only confirmed vecto r of malarial from 26 % to 7 3%, within 7 da ys and the parasites on Ni as (Indones ian M inistry of peak G i\\ int en sity was 44 5 gamctocytes/ul H ealth, u npub!. obs .). Anopheles sinensis (C I = 282-700Ipl) on day 7, was sign ificantly is a com mon, primarily zoop h iliclexophilic MALARIA ON NIAS ISLAND, SUJ\lA.TRA 459 an opheline of eastern Asia th at is rep ortedly artcsuna te may be man ageabl e, especially if refractory to most P. falcipanun infection s they could be reduced greatly by in-co untry (Somboon el al., 1994; Ron gsriyam et al., production ofArtemisia a111 wa. Even tho ugh 1998). The potential for P. uiuax trans most isolates of P. falcipa nun from Nias mission by Au. sinensis derives mainly fro m appear resistant to treatment with CQ an d the high de nsities th at population s of this SP, th e use of a combination of C Q and SP species can attain CRee et ol., 2001). T he might offer some ad vantage over the use present co llect ions of mosqu itoes on N ias of either C Q or SP as a first-line strategy we re limited in sampling time, season against unco mplicated, P.Jalciparum ma laria and locat ion and therefore probably biased. (Winsta nley, 2001). T ourists and trave llers ANopheles tcsselatus, An. sinensis and othe r to Nias arc advised always to usc bcdncts, freshwa ter-breeding an ophclincs undetected topi cal mosquito repellents and effec tive in the brief survey on coastal Nias may chemoprop hylaxis, to reduce the ir risk of be more ab un dant inland, whe re they may acquiring malaria on the island (and of play a more important role in malarial carrying multi-resistant strains to other transmi ssion . region s). In summary, active case de tection identified large numbers of asymptomatic infections and garnetocytae rnias, predo minantly amo ng ACKK'OW LEDGEl\1EI"TS . This work was su p young children, in the rura l population of ported by a Vete ran 's Administration grant southe rn, coa stal Nias. No evidence was (98-FRS-0273) , STEPF, 6.2 an d 6.3 of the found of increasing resistan ce to C Q in the U nited States M ilitary Infectious D iseases local P. cieox at this confirmed focus of Research Progr am , and th e D ep artmen t CQRPV but th e results of in-vivo testing of Defen se G lobal Emerging In fecti on s de mo nst rated poor therape utic an d clin ical System. The views and opinions expressed respon ses to SP in P. falciparuni. There was herein arc those of the authors and do not a high frequen cy of mutation s in the dhfr purpo rt to reflect or represe nt those of the gene that arc associated with pyrim ethamine U.S. Navy, th e U.S. Dep artment ofDefense , resistance in P. falciparum, but no sign of or the Indonesian Ministry of H ealth. T he selection pressure by sulfado xine in the dhps authors gra tefu lly acknowledge the assist gene. In add ition to its poor clin ical perform ance and sup port of Dr J\ 1. D avis of PT ance, 5 P ap peared to increase th e frequen cy Caltex Pacific Indon esia, Dr M . Kh airani of and intensity of garnetocytacmias, potentially T elu k D alarn , Nias, and me mbers of the heightening malaria transmission and disease Provincial H ealth Services ofNort h Su matra risk in the area. Alth ough CQ still affords and Nias. ~ I11is study was conducted in accord goo d treatment efficacy aga inst P. virax ance with the U.S. Navy's an d Republic on Nias, the use of 51' alone in the treat of Indonesia's regulations governing the ment of P. falcipanun on th e island sho uld protection of human subjects in med ical be stro ngly discouraged . SP sho uld only be research. American and Indonesian ethics used in combina tio n with a rapidly acting committees reviev..·ed and approved all the and garnetocytocid al arre misinin derivative procedures followed in this research. (Kumar and Zheng, 1990; Price el al., 1996). 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