Bulletin ofthe WorldHealth Organization, 63 (4): 731-737 (1985) © World Health Organization 1985

Prospective double-blind trial of two different doses ofmefloquine plus pyrimethamine-sulfadoxine comparedwith pyrimethamine-sulfadoxine alone in thetreatment of falciparum malaria

D. BOTERO M. RESTREPO,2 & A. MONTOYA2

This double-blind study is based on the treatment of 75 adult male patients sufferingfrom Plasmodium falciparum malaria in Medellin, Colombia, a city in which there is nomalaria transmission. The patients, who came from regions with high resistance toantimalarials, were divided into three groups receiving single-dose treatment asfollows: acombination of 280 mg mefloquine, 800 mg sulfadoxine and 40 mg pyrimethamine; acombination of420 mg mefloquine, 1200 mg sulfadoxine and 60 mgpyrimethamine; and acombination of1500mg sulfadoxineand 75 mgpyrimethamine. After treatment, follow-upexamination wasperformed dailyfor! week and then weeklyfor another3 weeks. The curerate in the mefloquine groups (within the study period of 28 days) was 100%, and in thethird group 75%o. Normal blood levels ofthe administered drugs werefound in 6patients ofthe third group who were not cured; they were subsequently cured with a single dose of1000 mg ofmefloquine. Drug tolerance was good and no toxic effects were demonstratedin blood and urine examinations. While the doses in the drug combinations (containingmefloquine) gave very good results, we would recommend a slightly higher dosecombination of mefloquine with sulfadoxine-pyrimethamine for the treatment offakciparum malaria in areas with a high prevalence of chloroquine resistance.

Owing to a variety of factors, malaria is at present aresurgent disease. The number of cases per year hasbeen estimated at over 300 million, and in Africaalone it is thought to be the cause of death of morethan 1 million people every year. One of the manyproblems involved with malaria is the widespreadresistance of Plasmodium falciparum to antimalarialdrugs (1). Although the chemotherapy of malaria isthe subject of many papers and other publications,practising physicians in the tropics may find themhard to obtain. Those with this difficulty should tryto consult the recent WHO monograph on thesubject(2).Chloroquine resistance in P.falciparum malaria

was well documented for the first time in 1961 in twopatients from the Magdalena valley in the north-eastern part of Colombia (3). The strain isolated from

' Professor of Parasitology, Faculty of Medicine, University ofAntioquia, P.O. Box 1226, Medellin, Colombia. Requests forreprints should be sent to this author.

2 State Health Central Laboratory and Corporation forBiological Investigations, Medellin, Colombia.

one of these patients was resistant to this drug inexperimentally induced malaria in humans (4).Several years later this resistance was also found incases from the country's Pacific coast (5), and hasrecently been confirmed both in vitro and in vivo in alarger number of cases from different parts ofColombia (6,7). In 1973 a review of the geographicaldistribution of resistance to 4-aminoquinolines citedother countries in Central and South America as wellas parts of south-east Asia (8).

Resistance of P.falciparum to combined pyri-methamine-sulfadoxine was initially seen in just afew cases in a study of experimentally inducedmalaria in Brazil (9). Recent publications from thatcountry have shown that, in cases from the Amazonregion, resistance to pyrimethamine-sulfadoxine wasas high as 630/ (10, 11). Similar findings were alsoreported from south-east Asia (12) and many othercountries, including Colombia (13). Resistance tochloroquine and pyrimethamine-sulfadoxine has alsobeen described in Africa (14) and in Papua NewGuinea (15).

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The widespread and increasing resistance ofP.falciparum to these drugs has necessitated thedevelopment of new antimalarials and more effectivedrug combinations. As a result, mefloquine orquinolinemethanol hydrochloride, which is related toquinine, was developed by the US Army AntimalarialDrug Program after screening more than 250 000possible drugs (16). With a mean half-life in thebloodstream of 21 days, mefloquine is active againstmultidrug-resistant strains of P.falciparum in singleoral doses. Since the initial phase I toleranceexperiments reported in 1975, clinical studies haveshown that this drug is well tolerated when takenorally, has a high radical cure rate in P.falciparuminfections, and displays dramatic suppressive(prophylactic) activity against this species and alsoagainst P. vivax (17).The combination of mefloquine with pyri-

methamine-sulfadoxine shows an additive bloodschizontocidal action and the development ofresistance was greatly delayed when tested inPlasmodium berghei (18). These findings support theinterest in clinical trials using these drugs for thetreatment of P.falciparum malaria. The tablet used inour study contained 140 mg mefloquine (base), 400mg sulfadoxine and 20 mg pyrimethamine. This isdifferent from the formulation currently beingdeveloped by Hoffmann-La Roche in collaborationwith WHO, which contains 250 mg mefloquine(base), 500 mg sulfadoxine and 25 mgpyrimethamine. The latter formulation was agreedupon following discussions between the company andWHO because it was considered that, with the lower-dose combination, a fully curative effect might not beobtained.

MATERIALS AND METHODS

This study was carried out from April 1982 toFebruary 1983 on 75 patients who had been infectedwith P.falciparum malaria in regions wherechloroquine-resistant strains were prevalent. Thesepatients were treated and followed up for 1 month inthe city of Medellin where natural transmission ofmalaria does not occur. The criteria for the selectionof patients were: males over 14 years of age,P.falciparum parasitaemia of more than 400trophozoites per mm3 with symptomatic disease, andno previous treatment with other antimalarials duringthe 4 weeks prior to entering the trial. All patients hada negative Dill-Glazko test for chloroquine in theurine. The patients were seen daily during the firstweek following treatment and then weekly for 3weeks, except for 7 cases who were followed up for aperiod of 1-3 weeks, 3 cases for 2 weeks, and 2 cases

for 1 week; only one patient dropped out.Age, weight, height, past history of allergic reac-

tions and concomitant associated illnesses, if present,were recorded for each patient at the initialexamination. All patients gave their verbal consent tobe included in the study. The following clinicalparameters were assessed before treatment and oneach visit: body temperature, blood pressure, pulserate, spleen palpation, liver palpation, and signs andsymptoms that could be attributed to malaria, such asheadache, vomiting and diarrhoea. The state ofconsciousness, presence or absence of chills, andcomplications as well as any adverse reactionsoccurring during the study were recorded, indicatingthe type, severity, date of onset and duration of thereaction. Any concomitant medication with otherdrugs was also recorded.The following laboratory parameters were assessed

before and after treatment: Hb level, RBC count,ESR, erythrocyte volume fraction, leukocyte countand differential count, platelet count, urineexamination, and serum determinations of bilirubin,creatinine, alkaline phosphatase, aspartate amino-transferase (SGOT) and alanine aminotransferase(SGPT).The trial was conducted as a prospective

randomized double-blind study in 3 parallel groups ofpatients, each receiving a single dose of 6 identical-looking tablets, which were swallowed with a glass ofwater in the presence of the investigator. Theantimalarials being investigated were: sulfadoxine(S), pyrimethamine (P) amd mefloquine (M), whichwere administered according to the following scheme:

Group 1 (25 cases): 2 tablets (corresponding to 280mgM + 800 mg S + 40 mg P) and 4 placebo tablets.Group 2 (26 cases): 3 tablets (corresponding to 420mgM + 1200 mg S + 60 mg P) and 3 placebo tablets.Group 3 (24 cases): 3 tablets (corresponding to 1500mg S + 75 mg P) and 3 placebo tablets.

The parasitological studies consisted of counts ofthe trophozoites and gametocytes of P.falciparumbefore starting treatment, followed by daily countsafter treatment for 7 days and then once a week for amaximum of 4 weeks. The presence of mixedinfections with P. vivax was recorded. Blood sampleswere collected before and 24 hours after treatment fordetermination of plasma concentrations of the 3tested drugs.

Treatment with 1000 mg of mefloquine wasreserved for those patients who were not cured withthe above treatments and in whom P.falciparumtrophozoites persisted in the blood and symptomswere present. The drug was given as a single dose andthese subjects were then followed up for the standardperiod of one month.

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RESULTS

Parasitological response

The initial parasite counts ranged from 425 to280 000 (mean ± SD = 33 300 59 177) in group 1,from 1849 to 300 000 (mean + SD = 28 433 ±

66 496) in group 2, and from 718 to 48 300 (mean ±

SD = 10 916 ± 13 064) in group 3. Considering the75 patients together (one later dropped out), we foundthat most cases presented low to moderate levels ofparasitaemia. Trophozoite counts were below10 000/mm3 in 42 cases, between 10 000 and100 000/mm3 in 30 cases, and above 100 000/mm3 in3 cases.

Clearance of the asexual blood forms ofP.falciparum was obtained within 5 days in groups 1

and 2 (no cases of parasitaemia after day 5 in group 1,after day 4 in group 2), and no cases of recrudescenceoccurred in any patients from these two groups. Themean parasite clearance time (± SD) was 3.0 ± 1.6days in group 1 and 2.8 ± 1.3 days in group 2. Six ofthe 24 patients in group 3 (sulfadoxine-pyrimetha-mine group) had to be treated with mefloquine (1000mg) in the period between days 3 and 8 because of anunsatisfactory parasitological response; in theremaining 18 patients, clearance of asexualparasitaemia was achieved within 4 days. Norecrudescences were observed, which means that thecure rate in the 50 cases treated with the twocombinations of mefloquine and pyrimethamine-sulfadoxine was 100% (within the study period of 28days), while the cure rate in the 24 patients treatedwith the standard dosage of pyrimethamine-sulfa-doxine was only 75%o. This difference between group1 and 2 on the one hand and group 3 is statisticallysignificant (Fisher's exact test, 2-tailed, P< 0.005).The gametocyte counts showed that 16 out of 23

patients in group 1, 11 out of 23 cases in group 2, and15 out of 16 cases in group 3 had a positive gametocytecount on days 12-16. On days 25-32, only 4 out of 20cases in group 1 and 3 out of 20 cases in group 2 stillhad a low gametocyte count. However, 8 out of 12patients in group 3 still had a positive and in somecases rather high gametocyte count.The plasma concentrations of pyrimethamine and

sulfadoxine, which were measured by means of aspecific and sensitive microbiological assay (19)before and 24 hours after the ingestion of these drugs,showed that in 6 patients who were not cured with thistreatment the levels were within the expected range,compared with those in volunteers. These findingsconfirm that the 6 patients had ingested and absorbedthe drugs and that they were genuinely resistant to thisdrug combination. This resistance was classified as RIin 1 case and RII in 5 cases.

Concomitant P. vivax infection was observed in 2patients during the 1 month follow-up period and in 6others after this period. These 8 patients remained in anon-malarious area and did not show P. vivax in thepre-treatment blood examination, which means thatthis form of malaria was due to relapses. All werecured with chloroquine and primaquine.

ClinicalfindingsNo impairment of consciousness was found in any

patient and the blood pressure showed no significantchanges at any time. The fever was cleared in 58% ofthe parasitologically cured cases in group 1 within 4days of treatment, in 641o in group 2, and in 69% ingroup 3. By day 7 all the patients were afebrile. Chillswere not observed after day 4 in any of the curedpatients. Headache and vomiting were commonsymptoms before treatment and did not occur afterthe third day of treatment. Diarrhoea occurred lessfrequently and persisted for a few days after therapy.Hepatomegaly (1-6 cm below the costal margin) waspresent before treatment in 17 patients from group 1,15 from group 2, and 16 from group 3. At the end ofthe follow-up period (days 25-32), liver enlargementwas recorded only three times in groups 1 and 2 andtwice in group 3. Splenomegaly, following theHackett classification, was found before treatment tobe between classes 1 and 4 in 24 cases from group 1, 24from group 2, and 22 from group 3; classes 1 and 2were the most common finding. At the end of thefollow-up period, slight splenomegaly (in most casesclass 1, i.e., palpable on deep inspiration) was foundin 20 patients from group 1, 20 from group 2, and 11from group 3.

Renal insufficiency was observed in only 2 patientswho showed a high parasitaemia of around 300 000per mm3 before treatment, and their symptomssubsided on day 2 after treatment. Concomitantbacterial infections were seen in 2 patients withpharyngitis and bronchopneumonia which werecured with penicillin.

Side-effects. Vomiting was the only symptom thatcould be regarded as a side-effect of treatment. Thiswas present with slight intensity in only three patients:two from group 1 and one from group 3.

Laboratory investigationsThe non-parasitological laboratory determinations

gave results in many cases that were outside thenormal range because of the malaria; no changes wereobserved that could have been due to the treatment.The main findings observed were low values for Hb,erythrocyte volume fraction, RBC counts and plateletcounts both before and after treatment. Raised valueswere seen before and after treatment for the ESR; 40

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D. BOTERO ET AL.

out of 73 cases showed raised total bilirubin valuesbefore treatment while only 5 patients still showed anincreased bilirubin on days 2-4 after treatment.Raised values for alkaline phosphatase, SGPT,SGOT and creatinine were observed in several casesbut the differences before and after treatment werenot significant. The main finding in urinalysis was ahigh frequency of proteinuria, which is to be expectedin symptomatic malaria.

DISCUSSION

A recent report on mefloquine discussed the safety,tolerance and efficacy of this drug (20). Advice wasgiven on how the development of resistance might beprevented by following various procedures, includingcombination of mefloquine with pyrimethamine-sulfadoxine. The effective dose for mefloquine, whenused alone, seems to be 1000 mg, administered as asingle dose (21). This dose was found to be effectiveby us in the 6 cases that did not respond topyrimethamine-sulfadoxine. We also found that thecombination of the three drugs was not effectiveagainst the tissue forms of P. vivax, a fact found alsofor mefloquine alone at the standard dose (21).

In our study, all 74 cases initially showed onlyP.falciparum, but after this infection was cured, 8cases with P. vivax appeared. Similar findings, withan even higher P. vivax relapse rate, were found inBrazil (22) where 21 out of 99 cases were positive forP. vivax after P.falciparum was cured. These findingssuggest a dominance of P.falciparum over P. vivaxwhen both parasites are concomitantly infecting thesame patient.A study in Brazil (22) recorded a 100%7o cure rate for

P.falciparum with 1000 mg of mefloquine, but only73% with the standard dose of pyrimethamine-sulfadoxine. In our study the cure rate with the latterwas 7507, and with the combination of 3 drugs it was100% (within the study period of 28 days); ourcombination contained only 280 mg of mefloquine ingroup 1 and 420 mg in group 2. A cure rate of only19.4% with pyrimethamine-sulfadoxine wasobtained in a study in Thailand, where mefloquineproduced much better results (23). In the samecountry, a comparative study of mefloquine (singledoses of 1500 mg) versus pyrimethamine-sulfadoxine(standard doses) showed a higher cure rate with theformer; also gastrointestinal side-effects (nausea,vomiting, diarrhoea) were more common withmefloquine (24). In P.falciparum cases in Burmatreated with mefloquine (in doses of 20 and 30 mg/kgof body weight for children and 750-1000 mg foradults) side-effects like nausea, giddiness andvomiting were noted in about 60% of the patients

(25). The side-effects attributable to mefloquine inour study were infrequent since vomiting occurred inonly 2 out of the 50 cases (in groups 1 and 2) who hadreceived combinations of the 3 drugs. This goodtolerance may be due to the smaller amounts ofmefloquine used in our study.

It is recognized that mefloquine in the therapeuticdose for P.falciparum is also effective against theblood forms of P. vivax (20) and P. malariae (26).This wide spectrum of efficacy against the three mainhuman malaria parasites is another factor in favourof this drug. At present, however, mefloquine maynot be required in regions where P.falciparum is stillhighly sensitive to chloroquine, as in Zambia (27).The prophylactic activity of mefloquine in drug-

resistant malaria has been studied in non-immunevolunteers, in whom patent parasitaemia wasprevented when they were exposed to infectedmosquitos two weeks after receiving a single dose of1 g of mefloquine (28). Similar studies revealed anabsence of parasitaemia during 60 days' follow-up ofvolunteers infected with a strain of P.falciparumresistant to chloroquine and pyrimethamine, afterreceiving different doses of mefloquine, with amaximum of 1000 mg every 4 weeks (29).A large comparative field trial carried out in

Thailand, where different weekly doses ofmefloquine and sulfadoxine-pyrimethamine wereadministered for 26 weeks to nearly 1000 persons,showed that mefloquine was more effective insuppressing both falciparum and vivaxparasitaemias. Both groups tolerated the drugs welland no toxic effects were recorded except forleukopenia in the second group. The recommendedweekly dose for a 50 kg individual was 180 mg ofmefloquine, which is equivalent to 250 mg for a 70 kgperson (30). The prophylactic use of mefloquine inchildren also gave excellent results with goodtolerance (31). Nevertheless, this drug should be usedonly in high-risk groups in special circumstances. Thepossibility of a combination with primaquine as agametocytocide and for the radical cure of vivaxinfections should be explored (20).The recommendation not to use mefloquine freely

and to combine it with other antimalarials is mostlybased on laboratory experiments which showed thatresistance appeared when repeated weekly passages.ofPlasmodium berghei in mice were maintained underincreasing drug pressure (32). Using the P. bergheisystem in rodents, several mefloquine-resistant lineshave been selected. All these lines lost their resistancequickly when passaged without drug pressure, andfortunately no definitely mefloquine-resistant strainsof P.falciparum have so far been obtained fromcultures in vitro (33). Experimental resistance to anantimalarial depends mainly on the size of theparasite population exposed to the drug pressure, but

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TREATMENT OF FALCIPARUM MALARIA

it seems the para-aminobenzoic acid content in thediet may influence this sensitivity by accelerating thedevelopment of resistance when the content of thisacid is high (34). The relation of these experiments todrug resistance in human malaria has not yet beenclarified.

Several clinical trials with combined mefloquineand sulfadoxine-pyrimethamine treatment forP.falciparum infections have been performed duringthe last few years. In one study in Brazil, using 750 mgof mefloquine combined with the standard dose of theother two drugs, there was a 100% cure rate; therewas good drug tolerance and rapid disappearance ofthe clinical symptoms (35). These results are similarto ours- 100% cure rate (S response) within thestudy period of 28 days, although our drugcombination included smaller amounts ofmefloquine. However we would not recommend

using such a low dose of mefloquine in our countrywhere there is already resistance to sulfadoxine-pyrimethamine. In order to avoid treatment failuresthat would favour the development of mefloquineresistance and the further spread of resistance tosulfadoxine-pyrimethamine, the dose of thecombination should be high enough to ensure a fullycurative effect in practically all cases. For this reason,a high-dose combination tablet containing 250 mgmefloquine, 500 mg sulfadoxine and 25 mgpyrimethamine has been developed (see above).Studies with this combination have already beenperformed; the results show that 2 to 3 of these higher-dose tablets are necessary to achieve a cure rate of100% in malaria regions where chloroquine-resistance is highly prevalent and where there is alsosome resistance to sulfadoxine-pyrimethamine (36).

ACKNOWLEDGEMENTS

We are grateful to Dr R. H. Leimer and Dr A. Duarte of F. Hoffmann-La Roche, Basle, Switzerland, for their efficientcontribution to the planning, developing and writing up of this study, and to the Malaria Eradication Programme, Medellin,Colombia, and the State Health Central Laboratory of the same city for their valuable cooperation.

RtSUMIt

ESSAI PROSPECTIF EN DOUBLE AVEUGLE DE DEUX DOSES DIFFtRENTES DE MtFLOQUINEPLUS PYRIMSTHAMINE-SULFADOXINE, PAR COMPARAISON AVEC LA SEULE PYRIMtTHAMINE-SULFADOXINE,

DANS LE TRAITEMENT DU PALUDISME A FALCIPARUM

La Colombie est un pays oui le paludisme pose unprobleme considerable, en partie du fait de l'augmentationde la pharmacoresistance de Plasmodiumfalciparum. Cetteetude avait pour objectif de tester 1'efficacite de deux dosesdifferentes de l'association mefloquine (M) plus sulfadoxine(S) et pyrim6thamine (P), par comparaison avec la doseclassique de S et de P seules, pour le traitement du paludismea falciparum. On a traite en double aveugle, a I'aide de dosesuniques, trois groupes randomises de 25, 26 et 24 hommesadultes malades, presentant une parasitemie de plus de 400trophozoites de P. falciparum par mm3, de la faconsuivante:Groupe 1 (25 cas): 2 comprimes (correspondant a

280 mgM + 800 mg S + 40 mg P) et 4comprimes de placebo

Groupe 2 (26 cas): 3 comprimes (correspondant a420 mg M+ 1200 mg S + 60 mg P) et3 comprimes de placebo

Groupe 3 (24 cas): 3 comprimes (correspondant a1500 mg S+75 mg P) et 3 compri-mes de placebo.

Tous ces malades provenaient de regions impaludees etont et traites et suivis dans la ville de Medellin, oui il n?y aaucune transmission du paludisme. La surveillance post-

therapeutique a dure 4 semaines, des examens cliniques etparasitologiques etant pratiques tous les jours au cours de lapremiere semaine, et une fois par semaine ensuite. On aprocede a des analyses de sang et d'urine avant et apres letraitement, en recueillant du plasma a cette occasion, afind'evaluer les- concentrations medicamenteuses dans lesang.Le nombre de trophoites 6tait inferieur a 10 000 par mm3

dans 42 cas, compris entre 10 000 et 100 000 par mm3 dans30 cas et superieur a 100 000 par mm3 dans 3 cas. On aobtenu une disparition de la parasitemie (formes asexuees)en 5 jours pour les groupes 1 et 2, sans recrudescence. Sur les24 malades suivis dans le groupe 3, 6 n'ont pas 6te gueris. Ladifference entre l'ensemble des groupes 1 et 2 (taux deguerison de 100%) et le groupe 3 (taux de gu6rison de 75%)est statistiquement significative. Chez les 6 malades nongu6ris, on a observe des concentrations normales desulfadoxine et de pyrimethamine apres le traitement, ce quiconfirme la resistance a ces medicaments. Cette resistanceetait du type RI dans un cas et du type R2 dans les 5 autres.Tolls ces patients ont ensuite e traites avec succes a l'aided'une dose unique de 1000 mg de m6floquine. Au cours dela periode de suivi ou juste apres on a releve la presenceconcomittante de P. vivax, chez 8 malades chez qui cette

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736 D. BOTERO ET AL.

espece n'avait pas e detectee avant le traitement.Les observations cliniques avant traitement correspon-

daient aux symptomes habituels du paludisme a P. falci-parum, sans alteration de la conscience. L'hyperthermie,qui est le symptome principal, a disparu chez la plupart desmalades gueris parasitologiquement dans les 4 jours suivantle traitement. Chez deux malades presentant uneinsuffisance renale avant le traitement, la parasitemie

elevee, d'environ 300 000 par mm3, a rapidement cede al'administration du medicament.

Les effets secondaires ont et rares, des vomissements(sans gravite) s'etant produits chez seulement deux maladesdu groupe I et un malade du groupe 3. Les analyses delaboratoire ont donne des resultats normaux pour despaludeens et n'ont pas r6v&lE de modifications importantesqu'on puisse attribuer a ces medicaments.

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