Studies of resistance to chloroquine, quinine, amodiaquine and mefloquine among Philippine strains of Plasmodium falciparum

TMNSA~~ONSor THB ROYALSocrurv OFTROPKXL&DICINE

AND

HYGIHNH(1985)79, 37-41

Studies of resistance to chloroquine, mefloquine among Philippine strains

37

quinine, amodiaquine and of Plasmodium fa/ciparum*

LLOYD L. SMRKOVSKI',RICHARD L. BUCK’, ALBERTOK. ALCANTARA’CAMILO S.RODRIGUEZ’ANDCESAR v. UYLANGc02 ‘Dept. of Immunology and Biochemistry, U.S. Naval Medical Research Unit No. 2, APO San Francisco, Ca. 96528, USA; 2San Lazaro Hospital, Manila, Philippines Abstract One hundred casesof slide-confirmed Plumodium falciparum malaria admitted to the San Laxaro Hospital, Manila, Philippines were screened for in vitro resistance to chloroquine, quinine, amodiaquine and mefloqnine using the microtechnique. 59 of the 100 primary parasite isolates produced schixonts, whereas the remaining 41 isolates did not. 51 of the 59 isolates tested were resistant in vitro to chloroquine and eight were sensitive. In contrast, three of the primary isolates were resistant to quinine, three showed resistanceto amodiaquine and four were mefloqnine-resistant. 43 of the strains judged chloroquine-resistant in vitro were fully in vitro sensitive to amodiaquitte, quinine and mefloqnine. One chloroquine-resistant isolate was also resistant to quinine alone. Three isolates that were resistant to chloroquine were also resistant to amodiaqnine. An additioy three were cross-resistant to chloroquine and mefloquine. A single isolate was found to he reststant to chloroquine, quinine and me&quine and another was cross-resistant to chloroquine, quinine and amodiaquine. All strains demonstrating in vitro resistanceto amodiaquine, quinine or mefloquine also showed in nirro resistance to chloroqnine. The parasites in 22 patients showed in vivo resistance to chloroquine therapy. 86% were of the Rl type, 9% were R2 and 5% R3. All 22 patients demonstrating in vivo resistance to chloroquine showed in vitro resistance. Itttroduction infections acquired in south-east Asia are at times not responsive to standard therapeutic regimens of chloroquine (25 mg/lcg) (HARINASUTA et al., 1982). These strains of the parasite, termed “chloroqnine resistant”, present a special problem to the clinician insofar as alternative choices of treatment. We have heen using the in vitro microtechnique (RIECKMAN et al., 1978) to detect parasite resistance, not only to chloroquine but also to amodiaquine, quinine and mefloquine in P. falciimmm casesat San Laxaro Hospital, Manila. The data acquired from these studies allows us to predict in vivo chloroauine resistancewithin 48 hours of diaanosis and provides baseline data on the sensitivity of local strajns of P. falciparum to the other three tmdanal compounds. The in vinu responseof local Y! falciparum strains to me&quine is of special interest since this drug is one of the newer antimalarials under investigation. The data derived from this four-drug, in vitro study is also important from a standpoint of multiple drug resistance. Phmodium

falcipancm

Materials aud Methods Patientsadmitted to the San Laxam Hospital?Manila, Republic of the Philippines with P. fulci~mummfections

ccdirmed by Giemsa-stainedthick and thin blood films were entered into the study. Blood (EDTA tubes,venepuucture)

and urine sampleswere collected from patients before treatment (25 mgflrg chlomquine base given over three days). The urine was tested for the presenceof sulpba compoundsandCamiuoquiuoliuesby useof the L&niu test

(BRUCECHWATT et al., 1981) and DiU-Ghuko test (LIL.. L~JVE~D& RARTW, 1970), respectively. Quantitation of malaria mrasites was done bv countinn the number of

parasites-per100leucocytesaud usiug a&udard couut of 8000 leucocytes/pl blood. Parasite counts were followed

daily or alternatedaysuntil negative.Patientswhoseparasite

couutsbecamenegstivewitbiu one weekbut whosein vitro test resulta showed chlomquine resistance were encouraged to stayia the hospitalfor at leasttwo weeks.All discharge-cl patientswererequestedto return for follow-up examinations weekly for one month. Culture medium used in the in win0 test for resistancewas prepared fresh weekly. RPM1 1640 (260mg) with Lglmamine wasaddedto 21 ml sterile distilled water (USP),

plus 1.0ml of HEPES(150m&nl)/Gentamiciu(100ugml). The medium was sterilized by passagethrough a -22 tux~ iilter (Millipore Corp.), 1 ml of 75% NaHcO, and2.5 ml of iy AB serumwasaddedand tbe me&urnwasstoredat Two hundredul of bloodwas added to sterile, plastic vials containing 1.8 ml of culture medium. The vials were gently

swirled to mix the contents.SO-plaliquots of the solution weredispensecl into wells of tlat-bottomtissueculture plates which bad been predosed with varying concentrations (pica-moles)of chlomquine (1, 2, 4, 5.7, 8, 16, 32), amodiaquiue(*25, .50, 1,2,4, 8, 16),quinine (4,8, 16,32, 64, 128,256)or mefl uiue (-5, 1, 2, 4, 5.7, 8, 16)by the technical staff of WH“8 , Geneva,Switzerland.The plates were incubated in a candle-jar at 37.5 to 38.5”c. Incubation time (maturation of rings to schizonts) was determined by periodic examination of duplicate cultures set up in au

tutdosedplate. Upon harvest,the supematautfluid from eachwell was removedand settled erytbrccytes(approx. 5 ul) used to make thick films. The t&us were air-dried for 30 miu. dipped into reagent-gradeacetone for one set to prevent sloughing (SMRKOVSKI& RODRIGUEZ,1981), stained with 2% Giemsa pH 7.2 and examined for schizonts (three or more nuclei present).A minimm of 200oil immentonfields were examined before a thick film was considered negative. Data in the tables of in tirro resistance or sensitivity are reported as the highest concentration of drug (p-moles/5 $ blood) in which schizogony occurred. Resistance is defined

Addressfor reprint requests: Publications office, U.S. Naval Medical ResearchUnit No. 2, APO San Francisco,CA %528, USA.

38

DRUM

RESISTANCE

0F

by WHO (Wemsdorfer1982-personalcommunication)as schizontsdeveloping(in wizro)in the presenceof 5-7p-moles of chloroquine, 4 p-molesof amodiaquine,64 p-molesof quinine or 16 p-molesof metloquine. All patientsshowingin viva resistanceto chloroquinewere treatedwith metakel&, a Fansidar-likesulphacompound. Results 100 casesof P. falciparum malaria admitted to the San Lazaro Hospital, Manila, Philippines were screened for in vitro resistance to chloroquine, quinine, amodiaquine, mefloquine and in vivo resistance to chloroquine only. 59 of the 100 pfimary parasite isolates in the in vitro resistance test, produced schizonts, whereas the remaining 41 isolates did not. Table I summarizes the in vivo and in vitro data for the 59 isolates that produced schizonts. The initial parasitaemia levels of these 59 isolates,ranges from 480 to over 300,000 asexual parasites/mm3biood and for those isolates that failed to produce schizonts in vitro, the parasitaemia levels were comparable. Periodic monitoring of the P. falciparum cultures from duplicate plates containing no antimalarial compounds showed that the production of detectable levels of schizonts was highly variable. Somecultures required more than 51 hours incubation (Nos. 317, 394 and 395), while others were positive for schizonts in 17 hours (Nos. 338, 357, 358). The urine-test data from all patients are presented in Table I and summarized in Fig. 1. 55% of the 100 patients studied submitted pre-treatment urine specimens; 38% were positive for 4-aminoouinolines and 62% were negative. All urines were negative for sulnha druzs. 57% of the 21 cultures initiated with puasites fr;m those persons with a positive urine test produced schizonts in vitro; 43% did not. Conversely, of the 34 cultures inoculated with malaria parasites from persons who showed no indication of prior intake of 4aminoquinolines (negative urine test), 82% underwent schizogony. 45 patients did not submit urine samplesand 19 (42%) of these showed schizont production in vitro. Table I also shows the in vitro data for each culture-positive isolate studied as well as the patient’s response to chlorwuine therapy and/or days of follow-up. For the sake of data-comparison, fhe in vitro drun data is summarized in Tables II and III. Table II Ghowsthat 14% of the 59 isolates tested were in vitro sensitive to chloroquine, whereas the remaininz 86% were resistant. In contrast. 95% of the isdates were sensitive to amodiaquine *and93% were sensitive to melloquine. 97% of the primary isolates were sensitive to quinine. Table III summarizes the data on multiple drug resistance. As previouslv discussed, 86% of the isolates tested were in w&o resistant to chloroquine. 42 of the strains (82%) iudaed chloroouine resistant were fully sensitive to’ amodiaquine,. quinine and mefloquine. On the other hand, one chloroquineresistant strain (2%) was also resistant to quinine alone. Three isolates (6%) were resistant to chloroquine and only amodiaquine, whereasone strain (2%) was cross-resistant to chloroquine, quinine and amodiaquine. Three parasite strains (6%) were resistant to chloroquine and mefloquine. One strain (2%) was resistant to chloroquine, quinine and mefloquine. All

PHILIPPINE

P. falciparum

strains, in vizro resistant to amodiaquine, quinine or mefloquine were also in vitro resistant to chloroquine. The parasites in 22 patients showed in vivo resistance to chloroquine therapy (Table I). 19 (86%) were of the Rl type, two (9%) were R2 and one (5%) was R3. The number of days for recrudescenceof the Rl clinical responders ranged from 5 to 28 days (median 21 days). All 22 patients demonstrating in viva resistance to chloroquine also showed in vitro resistance. The in vivo chloroquine response of the remaining patients is not known, either because of incomplete follow up (<28 days) or of the inability to exclude re-infection. Three patients (Nos. 338, 359, 374), who were in vitro chloroquine-resistant, remained free of detectable parasites at days 33, 38 and 35 days respectively. All me&elfin-treated patients responded by complete parasite clearancewithin three to five days. Discussion Chloroquine-resistant falciparum malaria was first observed in the Philinnines in 1969 (WERNSDORFER & KOUZNETSOV,19&i), but the first study using in vitro drug resistance testing was not reported until 1976 (VALERA & SHUTE, 1976). In this report they showed that in vitro resistance to chloroquine, by use of the macromethod (RIECKMANet al., 1968), correctIv predicted in vivo chloroquine resistancein 18 of 18 casesstudied. Since that time the macro-test has been redaced bv the micro-test (RIECKMAN, 1978) both of which have been shown to give comparable results (WERNSDORFER & KOUZNETSOV,1980). In this study, 100 primary isolates of P. falcipancm were tested for their in vitro responseto all four drugs, but only 59 isolates produced schizonts when placed in the in vitro environment. The reason for lack of growth of some primary parasite isolates is not clear but may be a result of previous intake of antimalarials by the patient before arriving in the hospital. 38% of the patients who submitted urine samples before treatment were test-positive for 4aminoquinolines. Furthermore, of those cultures inoculated -with parasitized blood from individuals with a positive nresumptive urine test, 43% failed to undergo schizogony, whereas, of those cultures containing parasitized blood from patients with a negative urine test, only 18% failed. These findings suggestan association between previous intake of antimalarials and subsequent failure of the malaria parasites to undergo schizogony in vizro. Even though the Dill-Glazko urine test is neither a quantitative nor a coniirmatory qualitative test for 4aminoquinolines, we believe that the test results may still be helpful in interpreting in vitro resistancedata. Our data show that in 21 patients were test-positive for 4whose urines aminoquinolines, 12 produced schizonts in vitro, of which nine were judged in vitro resistant to chloroauine and the remainina three sensitive. Even thouzb only one of the nine in ho resistant isolates was later confirmed to be in vivo resistant (No. 361) we believe that if in vitro schizogony occurs ‘in spite of a positive urine test, that the in vitro resistance pattern can still be used to predict in wivo resistance. Resistanceof P. falciparum parasitesto compounds other than chloroquine is well known, especially among those parasite populations endemic to southeast Asia (HARINASUTAet al., 1982). In vitro and in

39

L. L. SMRICOVSKI et al. Table

I-Assessment

of resimmcc

of 59 primary

isolates

of P. fahparum

to chloroquine,

amodiaquine,

quinine

I-w-t

ID No. 2% 297 290 300 302 303 304 305 317** 323** 331 332 333 335 336 337 338 339 341 344 345 350** 351 352 353 357 358 359 360 361 362 363 364 365 367 368 369 370 371 374 375 376 377 378 379 381 382 383 384 385 386 387 388 393 394** 395 3% 399 400

As.Xual Parasitacmia (/mm’ blood)’

2,4M)

l,@+O

12,000 10,000 WOO0 68,000 28,500 1,200 5,600 63,800 15,200 14,400

8WJ

25,600 5,120 l,@O 720 1,600 2,880 16,000 Wj‘+O l,@O 14,344 9,280 6,520

2,m

1,360

2,640 ‘+Q,m 85,400 24,000 6u@J 15,360

2,@33

2,800 2,240 2,960 56,160 27,600 45,360 9,040 68,000 480 36,400 5,840 6,320 15,520 1,500 107,000 18,500 16,640 24,800 14,960 21,600 5,440 300,000 2,720 11,680 19,200

CUlnUCS Negative for Schimnts 0-W 23

2 -

17, 51 16 16 18 20,49 26, 45 26, 45 21, 40 21 16 16 16 20, 44 21 24,48 24 24 21 15, 40 46, 67 19, 42, 70 -

and melloquine

drus

COlEClttMiOtl

(P-mzuvinp schizonts First Detected 41 41 41 46 46 48 66 64 40 21 21 21 42 25 25 17 23 41 40 24 19 72 19 19 17 17 48 68 68 21 21 63 40 40 40 40 64 41 41 72 24 42 42 43 49 21:30 21:30 43 40 21 46 21 64 92 % 24 20 20

Harvest Tie 0-W

64 64 46 69 67 67 53 71 64:30 45 40 40 40 47 30:30 30:30 65 51 47 45 43 48 72 44 44 42 41:30 49 72 72 40 40 87 45 64 41 41 66 43 42 73 25 44 44 67 67 zl 45 42 28 48 48 90 92 % 50 42 42

Urine-b

+ + N’s + NS NS NS NS NS NS NS NS NS NS NS + + + NS NS NS NS NS NS + -

+ A + +

chl. 5.7* 2 5.7* 4 8* 4 5.7’ 8’ 32’ 16* 8* 32* 32. 5.7’ 16+ 32* 16* 32* 8* 8* 8* 8’ 4 8* 5.7* 5.7* 32* 32* 4 8* 32* 32* 32* 32*. 4 4 5.7* 32* 5.7’ 16* 5.7’ 32’ 8’ 32’ 8’ 5.7* 16* 32* 16* 8* 8* 8’ 8* 5.7* 5.7* C 8’ 5.7* 32’

Amo. .5 C .25 .25 4* .5 1 1 2 1 1 2 1 1 2 1 1 1 1 1 1 .5 .25 .5 .25 .5 .5 2 .25 .5 4’ 2 4* 2 .25 c .25 .5 1 1 1 2 .5 2 1 .25 1 1 1 2 1 1 1 .5 .25 .25 1 1 1

Qui. 4 C 4 4 16 4 4 4 8 8 16 16 8 8 64* 32 16 32 4 32 32 8 4 16 16 4 8 16 t 16 16 8 8 i 4 C 32 8 4 8 16 16 16 8 32 16 16 64’ 8 16 16 16 8 C 32 32 32

Mef.

In viva’ (Days of follow-up)

2

(14)

(6) (3)

1 2 1 1 1 4 .5 1 1 2 .5 4 2 2 2 2 4 16* 4 2 2 2 1 5.7 2 .5 .5 4 2 4 2 1 1 2 .5 16* 4 2 5.7 1 2 4 : 2 4 16* 1 2 5.7 2 1 C 5.7 16* 4

(8) (15) (15) (3) Rl (28) Rl (8) Rl (19) Rl (18) Rl (16)

(22)

Rl (16) (1) R-3 (33)

(23)

Rl

(5) (13) (0) died R-2 (0) (0) (0) (19) (19)

(38)

(9) (21) (20) (22) (28) (28) (9) (0) (0) Rl (26)

Rl Rl Rl Rl Rl

(2) (35) Rl (22) (0) died (27) (27) (f-9 Rl (27) Rl (26) (13) (3) (13) Rl (16) (35) (4) Rl (20) R2 (0) Rl (27) (30)

(2)

’ based on 8,000 leucocyteshm’ blood. b Urine test for 4-aminoquinolincs (DilLGlasko test); NS = none submitted. c Observed patient response &a chloroquine therapy (25 mgikg base), number in parentheses is the number of days of follow-up with no parasitaemia detected. R-values a presented followed by the number of days to recrudescence (WHO, 1973). l Schizonts developing in wells containing these drug concentrations indicate resistance (Wernsdorfer, 1982 personal communication). **Patients whose primary isolates did not grow in vine when cultured the first time. All four patients subsequently showed in uiw resistance. The in vitro and urine data presented is that from the recrudesces&g parasite population. The data on urine test results and negative culture results of the initial parasite population, of these four patients, is included in Fig. 1.

40

DRUG

r

Urine Positive (21 = 39%)

Urines submitted (BB)TDili-Glazko

r I

IL

I

100 cases

RESISTANCE

r

P. fakiparum

OF PHILIWINE

Positive 112 = 57%)

Table III-Summary of multiple in vitro drug resistant patterns among 59 isolates of P. falcipanim

I GrowthC . in vitro

No. isolates

I. Response to chloroquine a. Sensitive b. Resistant

L

5;

Negative (9 = 43%)

59

II. Cross-resistant pattern a. Chioroquine only b. Chloroquine + only quinine c. Chloroquine + only amodiaquine d. Chloroquine + only mefloquine e. Chlortxprine + quinine + amodiaquine f. ~hl;l~t$n,’ quinine

testb

Urine Negative (34 = 82%) -

r

Positive (28 = 82%)

I G;owth --in vitro

P. falciparum”

L

Negative (8 = 18%)

r

Positive (19 = 42%)

-

Urines not Submitted 1451

* Slide-confirmed b Urine test for 4-aminoquinolines-submitted treatment. c As determined by maturation of ring-stage schizonts in the absence of drug pressure. Fig. 1. Effecf of urine positivity teat) on in via0 slszogony.

Table

1.

2.

3.

4.

II-Zn

for Caminoquino~

prior to forms to

(Dill-Gluko

viwo response of 59 isolates of P. fakipatvm to four antimalarial

Control :

I!

% Total Cumulative % Amodiaquine No. isolates % Total Cumulative % Quinine

2 Control 2

-

4 *25 50 10 10

; coYol

::

x Control :

42

(82)

1

(2)

3

(6)

3

(6)

1

(2)

1

(2) ww

compounds

Highest concentration of drug (p-moles) allowing schizogony 2 4 5*7* 8* 16* : 6 :3 :4 6

Chloroquine No. isolates

No.Total isolates % Cumulative % Meflqsuine Jp&te

(86)

t&o studies of multiple drug resistance are few in number and are of obvious importance. In this study, it was noted that of 5 1 P. fakipatwn isolates judged in viwo resistant to chloroquine, 15% (9/59) showed in viuo cross-resistanceto one or more of the other drugs tested. This finding is not surprising in view of the similarities in chemical structure shared by these four compounds. If we assumethat the in niwo data for the compounds other than chloroquine accurately predicts in wivo resistance, then the contrasting patterns of multiple resistance observed are of interest. The use of m&oquine in the Philippines, unlike chloroquine, has been minimal, confined to two clinical trials involving less than 100persons (Drs. Rivera and Uylangco-personal communication, 1982). Thus,

Negative (28 = 58%)

:; 1:

(14)

(100)

51

Growth --in vitro

L

w>

10 14 l,“/ ii 80

8

;: g *5 1 2: : ::

36 4* 3

;:

63 i

1Ci q

ii 16* 0

32* 16 12

=

:;

32

64*

128*

256*

f2

1; 97

: 100 8 8

2 16* 4

2 -

4 :s

5.7 4

Cumulative % 5 13 35 zz 86 9’; 93 *Schizont formation in the presence of these drug concentrations suggests resistance.

14

L.

L.

SMRKOVSKI

the presenceof mefloquine resistance due to selection pressure and/or induction seemsunlikely indeed but, as suggested by PETERS et al. (1977), may be related to the chloroquine resistance pattern observed among these strains tested. Our data on mefloquine resistance is in contrast to the observations of LOPEZANTUNANO& WERNSDORFER (1979). They reported that chloroquine-resistant P. f&lcipanCm stiain~ from Brazil and Colombia. South America were six to eight times as sensitive to mefloquine as to chloroq&e when tested in vitm (micro-technique). Additionally, Doberstvn and co-workers have reported on the successful use of mefloquine in the treatment of chloroouine-resistant falcinarum malaria in Thailand (DOBE&YN et al., 1979); although others (HALL et al., 1977) have reported that mefloquine plus quinine appears to be superior to mefloquine alone. Further studies of multiple drug resistance are needed before these findings can be fully interpreted. Of equal importance, the data presented herein may also provide baseline data for the monitoring of possible shifts in the existing patterns of resistance. The data presented on multiple drug resistance suggest a relationship between resistance to chloroquine and the other three compounds tested. This hypothesis is supported by the observation that all isolates found resistant to amodiaquaine, quinine or mefloquine were cross-resistantto chloroquine. These observations suggest that a genetic aberration giving rise to chloroquine resistance may also trigger or regulate concomitant expressions of resistance to other distantly related compounds. This could explain how multiple drug resistance might arise in the absenceof multiple drug pressure as is seen here in the Philippines where among the four compounds tested in vitro, only chloroquine is widely used. It should be noted from the data reported that some malaria parasite populations, derived as primary isolates, will not undergo detectable schizogony in vitro within the first 24 to 30 hours, incubation. The periodic monitoring of a small number of the cultured malaria strains reported herein confirmed our earlier findings (SMRKOVSKI et al., 1983) that some cultures will not produce schizonts unless incubated for an extended period (45 to 70 hours). The reasonsfor this are unclear and do not appear to be associatedwith initial parasite density, urine test results or size of the parasite when first placed in culture. The initial size of the ringstage parasites is often helpful in predicting incubation time but cannot be relied upon for some StNiIlS.

The breakdown of in vivo resistance by Rl , R2 and R3 (86%, 9% and 5% respectively) can be compared to that reuorted in an earlier study at San Lazaro Hospital where it was found that 62% of the in vivo resistant caseswere Rl, 28% R2, and 10% R3 (BUCK et al., 1982). Acknowledgements We wish to gratefully acknowledge the technical assistance of the staff of the World Health Organization, Geneva, Switzerland for providing the drugdosed plates used in this study. We also wish to thank Dr. Nunilon Sy, and Patricia Ma&gay for their technical assistance. The authors also wish to thank Dr. John Cross for his valuable assistancein the review of this manuscript. This study was supported through funds provided by the Philippine Ministry of Health and the Naval Medical

41

t?t id.

Research and Development Command, Navy Department for Work Unit No. MRO41.09.01.0083. The opinions and assertionscontained herein are those of the authors and are not to be construed as official or as reflecting the views of the Philippines Ministry of Health, the U.S. Navy Department or the Naval Service at large. References Bruce-Chwatt, L. J., Black, R. H., Canlield, C. J., Clyde, D. F. & Peters, W. (1981). ChemotAempyof Mahia (2nd edit.) Geneva: World Health Organization. Buck, R. L., Alcantara, A. K., Uylangco, C. V. & Cross, J. H. (1982). Malaria at San Lazam Hospital, Manila, Philippines, 1979-1981. American Jotma of Tropical Medicine and Hy@ne, 32,. 212-216. DobcmpI E. B.,.Phmtuyothm, P., Noeypatimonoh, S. & mkta&amlom, C. (1979). Smgledose therapy of

falciparummalariawith mefloquineor pyrimethaminesulfadoxine.B&tin of the World Health Organization,

57, 275-279. Hallil., Dobemtyn, E. .B., Karnchanachetanee, C., ssmruqkc, S., Latxuthat, B., Pearhnan, E. J., Lampe, R. M., Miller, C. F. & Pbintuyothin, P. (1977). Sequential treatment with quinine and mefloquine or quinine and pyremethamine-sulfadoxine for falciparum mshuis. Britssh Medical Joumal, i, 1626-1628. Harinasuta, T., Dixon, K. E., Warrell, D. A. & Doberstyn, E. B. (1982). SEAMEO-TROPMED Scientific Group Meeting: Recent Advances in Mahuia with Special Reference to Southeast Asia. Bangkok: SEAMEOTROPMED Project.

Lilijveld, J. & Ramnan, H. (1970).The eosincolour testof Dii and Glazko: a simple field test to detect chloroquine in urine. Bulktin of the World Health Organization, 42, 477-479.

F. J. & Wemsdorfer, W. H. (1979). In ino response of chloroquine-resistant Plasmodiumfakiparum to meBoquine. Bulktin of the World Health Orpniaation, 57, 663-665. Peters, W., Howells, R. E., Portus, J. H., Robinson, B. L., Thomas, S. C. & Warhurst, D. C. (1977). The chemotherapy of rodent malaria, XXVII: SNdk on me& quine (WR 142,490). Annals of Tropical Medicine and Parasitology, 71, 407418. Ricckmsn, K. H., McNamara, J. V., F&her, H., Stocket-t, T. A., Carson, P. E. & Powell, R. D. (1968). Effects chloroquine, quinine, and cycloguanil upon the maturation of asexual erythrocytic forms of two strains of Plurmodium falcipanun in vitw. American Journal of Tropical Medicine and Hygiene, 17, 661-671. Ricckmm, K. H. (1978). Drug sensitivity of Plasm&urn falciparum an in vifro microtechnique, Lancet, i, 22. Smrkovski, L. L. & Rodriguez, C. S. (1981). Malaria blood culture: a rapid lixation method to prevent sloughing of the thick 6lm. Transactionsof theRoyal Societyof Tropical Medicine and Hygiene, 75, 901902. Smrkovski, L. L., Alcantara, A. A., Buck, R. L., Sy, N. E., Rodriguez, C. S., Macalagay, P. S. & Uylangco, C. V. (1983). Chloroquine resistant Plumodium falciparum: Effect of rabbit serum and incubation time on the in oirro (microtechnique) prediction of in wivo resistance. SoutheastAsianJoumal of Tropical Medicine and Public Health, kyCZ-hNnatIO,

14, 228-234.

Vale& C. V. & Shute, G. T. (1976). Preliminary studies on the responseof Plusmodiumfalciparum to chloroquine in the Philippines with the in oitro technique. Bulletin of the World Health Organization, St, 391-398. Wemsdorfer, W. H. (1980). Fteld evaluation of drug resistance -..A .._” in malaria; in vitro microtest. Acta Tropica, 37, LLL-LL/.

Wemsdorfer, W. H. & Kouznetsov, R. L. (1980). Drug resistant malaria-occurrence, control, and surveillance. Bulktin of the World Health O~anization, 58, 341-352. Accepted

for publication 4th February, 1984.