Clones of different sensitivities in drug-resistant isolates of Plasmodium falciparum

Bulletin ofthe World Health Organization, 61(4): 709 - 712 (1983) © World Health Organization 1983 Clones of different sensitivities in drug-resistant isolates of Plasmodium falckiarum SODSRI THAITHONG 1 Clones of two Thai isolates of Plasmodium falciparum were prepared and examined for variations in drug susceptibility and electrophoreticproperties ofenzymes. Both isolates werefound to include mixtures ofgenetically distinct parasites. Ofparticular significance was thefinding that one isolate (T9), which on initial testing was resistant to chloroquine, pyrimethamine and sulfadoxine-pyrimethamine, yielded a clone of parasites markedly more sensitive to these three drugs, whilefive other clones resembled the original isolate in drug susceptibility. The spread to Plasmodium falciparum of resist- ance to currently used antimalarial drugs is a major constraint to control programmes in many parts of the world today. Following the confirmation of reports of resistance of this parasite to sulfadoxine- pyrimethamine combinations in south-east Asia and South America (1, 2), resistance to quinine has been reported in south-east Asia (3). In addition, chloro- quine resistance has spread further in this region and in South America and its appearance in local popu- lations in at least six countries of East Africa was confirmed in 1982 (4). However, little is known about the genetics of drug resistance and how it spreads in a natural population. Naturally occurring P.fal- ciparum always appears to be a mixture of popu- lations, i.e., including parasites that differ in a considerable number of parameters (2). Thus it can be suggested from drug-response curves that a natural isolate may be heterogenous in its sensitivity to a particular drug. The present paper confirms this hy- pothesis and shows that two isolates of P.falciparum from Thailand, which were shown to be resistant to chloroquine, contained parasites that were both resistant and sensitive to the drug. MATERIALS AND METHODS Ten clones, as defined by the presence of a single isoenzyme type of each enzyme studied, were established from the two isolates, PB, and T9 by a method based on limiting dilutions (5). PB1 was obtained from a patient in Phra Phutthabat and To from a patient in Ban Mae Sot, near Tak. The isolates were first characterized by enzyme electrophoresis (6) and then maintained in continuous culture for several 1 Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 5, Thailand. months using the method of Trager& Jensen (7). This was followed by cryopreservation in liquid nitrogen. At the time of isolation, PB, contained two forms of adenosine deaminase (ADA-I and ADA-II) and T9 contained the identical isoenzymes of ADA and two forms of glucose-6-phosphate isomerase (GPI-I and GPI-II), all of which appeared to be present in equal amounts. This suggests that both isolates were mix- tures of clones, as indicated by previous studies con- ducted by Rosario (5). These isoenzymes were observed in the isolates during the whole period of study although, during cultivation and cryopreser- vation, the staining intensity of the respective bands varied. This may indicate that during laboratory maintenance certain clones may be selectively favoured or lost. This observation has been con- firmed by the reaction of the two isolates to strain- specific monoclonal antibodies (McBride, personal communication). The two isolates were thawed from liquid nitrogen and maintained in continuous culture for one month before cloning of the isolates was initiated. Prior to cloning, enzyme electrophoresis indicated that PB1 had similar isoenzyme activities to those present at the time of isolation, whereas T9 showed similar activities of ADA-I and GPI-I but weak activity for GPI-II and ADA-II. Drug sensi- tivity testing was also carried out on the isolate prior to cloning. Cloning was initiated at a parasitaemia of 0.5%o, in which 12-18%o of the infected cells had two or more parasites. The technique used was that described by Rosario (5). RESULTS AND DISCUSSION Six clones were established from T9 and four from PB,. Each clone was tested for susceptibility to chloroquine and pyrimethamine by the method of 4331 -709 S. THAITHONG o ;o o X X X (DCD CD - 000 0 0 0' -, -, o oo _ _ 0 x CD 0 x CD 0 x CD 0 x CD CN4 0 x CD 0e o x UC C14 0 0 x C 0 0 x C- 0 x C 0L X X _ v- O O X _- _- _- X T- LCD 10 so 0 00 Ox0 CD4 l0 0 0 0 O__ o__ 4. 4. o s I 0 0'-00 _- _ X q- r- V) 0.m :m I 0 710 00- x x x x CD C CDCD0 0 0 x CD I.. 0 x CD 0 0 x x CD U) 0 x CD) 0 x CD) co OL C a; M 3 -' .R 0 -0E C- E a) c .E m a) 3 a) E ._ 0 c C C ._ 0 E : c .C 0 0. o 0 E .: -5Z E 0 cm x .0 C 0 C C 0 C. 0 .0:t C E E ._ E 0) - x .0 0 c C._ 0 C._>- E C ay a) E C. w~ 0 0 x CD 0 x LO U) 0 r- x CD 10o Is a )~- .0 .L C ..a c - .T o ,' E ).E E ._ c E m 0 c ._ aC C 30 .C a) 0 E a) C .5 0 ,o o 0 -c 0 E w< > iCLN> CW C 0 0 0 x LI) 00 x C) 0 x LX 0 x LO 0 x LO 0 x LC) 0 X ee CX 0 x Lo 0 x V) 00 x LO 2.Z m S) CC) O.-0 0 4-5 n .U WI- 0 Un a) C 0 Z 0 U) a) E ._ N C 4)0 'A C D0 C.) U) 0 a) a) I- ". a) C._4-0 a) 4-5 m .0 U1)d a) C 0 ZC1.) 0 U) a) E N C a) 0 U) 'a C._ m .t ._ a) .) Q (A U) 0) a) 6 C .C 0 a1) 0 .-- a) .0 a1) .0 C .2_ .6 0C 0 0i C) ._ C 0 i 0 CB .2 6 DRUG-RESISTANT ISOLATES OF P. FALCIPARUM Thaithong & Beale (8), to amodiaquine, quinine and mefloquine by the methods described by Thaithong et al. (9), and to sulfadoxine-pyrimethamine (Fansidar) using RPMI 1640 medium minus 4-aminobenzoic acid (Thaithong et al., unpublished). In addition, each clone was characterized by gel electrophoresis for ADA and GPI and for peptidase (PEP) in the case of clones from the isolate T9. The results (Tables 1 and 2) indicate that, prior to cloning, PBI and T9 were resistant to chloroquine, amodiaquine and pyrimethamine, but sensitive to quinine and mefloquine. They differed, however, in their response to sulfadoxine-pyrimethamine, PB1 being ten times more sensitive to the drug combination. Following cloning, Tg was shown to contain one clone (T9/107) which was sensitive to chloroquine, pyrimethamine and sulfadoxine-pyri- methamine, four which were resistant to all three of these drugs, and one (T9/98) which had an inter- mediate response to chloroquine but was resistant to pyrimethamine and sulfadoxine - pyrimethamine. Minor differences in the response of the individual clones to amodiaquine, quinine and mefloquine were also observed, but their significance is unknown. All four clones prepared from PB1 showed similar patterns of drug sensitivity to the parent isolate, with the exception of PB1/4 which was two times more sensitive to chloroquine. All were sensitive to quinine and to mefloquine. Further investigations will be made to determine the relationship between drug sensitivity and isoenzyme patterns in particular clones. Although the number of clones studied so far has been small, it is clear from the results presented that an isolate of P.falciparum, which has been shown to be resistant to a particular drug by in vivo or in vitro studies, may contain parasites of varying suscepti- bility including complete sensitivity to this drug. It is of interest to note that clone T9/107, which was sensi- tive to chloroquine, pyrimethamine and sulfa- doxine-pyrimethamine, was shown to grow in vitro at a slower rate than that observed in chloroquine- resistant clones. This observation has been made on other chloroquine-sensitive clones not reported here, and appears to correlate with the observations made by Rosario et al. (10) that chloroquine-resistant parasites of P. chabaudi had a selective advantage over those which were chloroquine-sensitive. Differences in the clones have also been observed in their reaction to strain-specific monoclonal antibodies, the results of which will be reported elsewhere. ACKNOWLEDGEMENTS This study received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases and British Petroleum Trading Limited. The kind assistance of Professor G. H. Beale and Dr D. Walliker during this study is gratefully acknowledged. RESUME CLONES DE SENSIBILITE DIFFtRENTE DANS DES ISOLEMENTS PHARMACORESISTANTS DE PLASMODIUM FALCIPARUM Deux isolements de Plasmodiumfalciparum provenant de Thailande (PB1 venant de Phra Phutthabat, et T9 venant de Ban Mae Sot, pres de Tak) ont et etudies pour verifier I'heterogeneite des parasites sur le plan de la pharmaco- sensibilite et des proprietes electrophoretiques des enzymes. L'isolement PB1 etait a l'origine dix fois plus sensible a la sulfadoxine-pyrimethamine que Ts, et l'on constatait egale- ment de petites differences dans la sensibilite a l'amodia- quine et a la quinine. En revanche, la reponse a la chloro- quine, a la mefloquine et a la pyrimethamine etait la meme dans les deux isolements. On a trouve dans l'isolement PB1 deux variants de l'enzyme adenosine-desaminase (ADA-I et ADA-Il) et dans l'isolement T9, outre les deux variants sus- mentionnes, deux variants de l'enzyme glucose-phosphate- isornerase (GPI-I et GPI-I1I). Des clones ont et prepares par la methode des dilutions limitantes: quatre A partir de PB1 et six A partir de T9. Chaque clone a et etudie sur le plan de la sensibilite aux medicaments et caracterise par electrophorese des enzymes. Les clones provenant de PB1 n'ont guere montre de diff&- rences dans leur sensibilite A tous les medicaments essayes. L'un des clones possedait l'enzyme type ADA-I, les trois autres ayant ADA-II. En ce qui concerne les clones provenant de T9, l'un (T/1107) etait nettement plus sensible A la chloroquine, A la pyrimethamine et A la sulfadoxine-pyrimethamine que les cinq autres clones ou que les isolements originels non clones. Parmi les clones de T9, on a trouve differentes combinaisons des variants des enzymes ADA et GPI, ainsi que des variants d'une troisieme enzyme, la peptidase (PEP), qui ont ete notes au cours de la derniere partie du travail. Il en est conclu qu'un isolement de P.falciparum qui, aux epreuves in vivo et in vitro, s'est revele resistant A un ou plu- sieurs medicaments peut neanmoins contenir des clones de parasites presentant A l'egard des memes medicaments des degres de sensibilite variables, y compris une sensibilite complete. 711 712 S. THAITHONG REFERENCES 1. PINICHPONGSE, S. ET AL. An evaluation of five regimens for the outpatient therapy of falciparum malaria in Thailand 1980-81. Bulletin ofthe World Health Organ- ization, 60: 907-912 (1982). 2. WORLD HEALTH ORGANIZATION. Malaria parasite strain characterization, cryopreservation and banking of iso- lates: a WHO Memorandum. Bulletin of the World Health Organization, 59: 537-548 (1981). 3. REACHER, M. ET AL. Drug therapy for Plasmodium falciparum malaria resistant to pyrimethamine- sulfadoxine (Fansidar). A study of alternate regimens in Eastern Thailand, 1980. Lancet, 2: 1066-1068 (1981). 4. WORLD HEALTH ORGANIZATION. Malaria chemo- prophylaxis. Weekly epidemiological record, 57: (No. 49): 381-384 (1982). 5. ROSARIO, V. Cloning of naturally-occurring mixed infections of malaria parasites. Science, 212: 1037-1038 (1981). 6. THAITHONG, S. ET AL. Enzyme typing of some isolates of Plasmodium falciparum from Thailand. Transactions ofthe Royal Society of Tropical Medicine andHygiene, 75: 268-276 (1981). 7. TRAGER, W. & JENSEN, J. B. Human malaria parasites in continuous culture. Science, 193: 673-675 (1976). 8. THAITHONG, S. & BEALE, G. H. Resistance of ten iso- lates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitro test. Transactions of the Royal Society of Tropical Medicine and Hygiene, 75: 271-273 (1981). 9. THAITHONG, S. ET AL. Susceptibility of Plasmodiumfal- ciparum to five drugs: an in vitro study of isolates mainly from Thailand. Transactions qf the Royal Society of Tropical Medicine and Hygiene, 77 (2): 228- 231 (1983). 10. ROSARIO, V. ET AL. Persistance of drug-resistant malaria parasites. Lancet, 1: 185-187 (1978),