Use of the in vitro microtechnique for the assessment of drug sensitivity of Plasmodium falciparum in Sennar, Sudan

Bulletin of the World Health Organization, SS (5): 785-789 (1980) Use of the in vitro microtechnique for the assessment of drug sensitivity of Plasmodium falciparum in Sennar, Sudan R. L. KOUZNETSOV,1 W. ROONEY,2 W. H. WERNSDORFER,3 A. A. EL GADDAL,4 D. PAYNE, 5 & R. E. ABDALLA 6 In 1978, studies on the chloroquine sensitivity ofPlasmodium falciparum were carried out in the district ofSennar, Sudan. The results ofthe in vivo testsshowedparasites resistant at the RI level only, but the mean clearance time oftrophozoitesfrom the blood was higher than for strainsfound in many other areas of tropical Africa. The in vitro tests, using the microtechnique, indicated a lower sensitivity to chloroquine in the local P. falciparum isolates than in those of most other African countries. However, similar results have been reportedfrom Ethiopia. The chloroquine sensitivity ofP. falciparumfrom Sennarisclose to the critical level of resistance. The in vitro microtechnique was also used to test for the sensitivity to Dabequin, 4-aminobenzo-quinoline, and was generallyfound to be a suitable and reproducible method, with a greater potential than the standard macro method. At parasite densities of over 100 000 asexual parasites per microlitre of blood the effect of a given concentration ofchloroquine was related to theparasite density owing to the selective uptake of the compound by the parasitized cells. In November 1978, at the end of the main malaria transmission season, investigations were carried out in Sennar District, Sudan, in order to: (a) establish the level of in vivo and in vitro sensi- tivity of the local strains ofPlasmodiumfalciparum to chloroquine; (b) obtain baseline data on the in vitro sensitivity of P.falciparum to new drugs; and (c) evaluate the potential of the recently developed in vitro microtechnique under field conditions. The study area had originally been mesoendemic for P.fakciparum, with localized hyperendemic areas (1). Between 1958 and 1971, extensive spraying operations with residual insecticides had virtually eliminated I Medical Officer, Programming and Training, Malaria Action Programme, World Health Organization, Geneva, Switzerland. Present address: Marcinovskij Institute of Medical Parasitology and Tropical Medicine, Moscow G435, USSR. 2 Laboratory specialist, WHO Regional Office for South-east Asia, New Delhi, India. 3 Chief, Research and Technical Intelligence, Malaria Action Programme, World Health Organization, 1211 Geneva 27, Switzerland. Requests for reprints should be addressed to this author. 4 Director-General, International Health Affairs (former Director, Malaria Control Division), Ministry of Health of Sudan, Khartoum, Sudan. 5 Technical Officer, Research and Technical Intelligence, Malaria Action Programme, World Health Organization, Geneva, Switzerland. 6 Department of Medical Microbiology and Parasitology, Faculty of Medicine, Khartoum, Sudan. indigenous malaria, but an interruption of major protective measures between 1971 and 1975 had led to the recurrence of focal epidemics in a number of villages. Preliminary studies Surveys conducted in Sennar Town and five neigh- bouring villages showed a low parasite prevalence. Sennar Town and two of the villages had no positive results while parasite rates in the remaining three villages were below 5 %. A fever-case survey revealed 131 slide-positive malaria cases out of 1515 patients (8.6%). Most of the patients were suffering from acute malaria, having contracted the infection outside Sennar District. Splenomegaly and gametocytaemia were rare, indicating that the disease was of recent onset. Individual case histories showed that the patients usually reported for treatment within a few days or even hours of the onset of symptoms. MATERIALS AND METHODS Selection ofpatients The subjects included in this study had attended the outpatient departments of Sennar Hospital, Sennar 4004 785- 786 R. L. KOUZNETSOV ET AL. Health Centre, or Juma Dispensary and had been found to be infected with P. falciparum. They had parasite densities between 1000 and 150 000 para- sites/lu of blood, as shown by the Giemsa-stained thick blood film. Patients with mixed infections, severe clinical manifestations, recent history of treat- ment, and urine tests positive for 4-aminoquinolines were excluded from the study. The selected patients were aged between 3 and 77 years. Method The in vivo response of P.falciparum to chloroquine was determined by means of the WHO Standard 7-day Field Test (2). A modification of the microtechnique described by Rieckmann et al. (3) was used for the in vitro assessment of the response of P.falciparum to chloroquine, Dabequin (4-aminobenzo-quinoline-a recently developed drug from the USSR) and mefloquine. The sealed microplates used for the testa contained 0.1 mg of dried ethylenediaminetetraacetic acid (EDTA) as an anticoagulant in well 1. Wells 2 and 3 were untreated to serve as controls, and wells 4-12 were predosed with 1.0, 2.0, 4.0, 5.0, 6.0, 8.0, 12.0, 16.0, and 32.0 pmol of chloroquine or Dabequin, or with 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, and 16.0 pmol of mefloquine, respectively. Blood was taken with a 100 Il capillary and placed in well 1. After resealing the well, the plate was agitated slightly to dissolve the anticoagulant. The plate was then taken to the laboratory within two hours, where wells 2-12 were each dosed with 50 ,l of growth medium (RPMI 1640, HEPES buffer, sodium bicarbonate, gentamicin) (3). The plate was again agitated briefly to allow the drugs to dissolve. Aliquots of 5 MA of blood fro.n well 1 were then added to wells 2-12. The plate was then covered, placed in a candle jar, and incubated, following the method of Trager & Jensen (4). The plates were removed after 24-26 hours and thick films prepared from the sediment of wells 2-12. After Giemsa staining, schizont counts were read and expressed on the basis of 200 asexual parasites. The growth rates in the drug wells were expressed as a percentage of the mean growth in the two control wells. RESULTS The in vivo response of P.falciparum to chloroquine was assessed in 26 subjects with counts of 1300-150 000 parasites per 14l of blood. The parasite density index of the group (5) was 8.78. The mean a Falcon No. 3040, Becton Dickinson, Zurich, Switzerland. parasite clearance time was 2.5 days with a range of 1-4 days. There was no difference in the response of persons with heavy or light parasitaemia. In general, the clinical symptoms responded more rapidly than the parasitaemia. Blood samples from 31 patients were tested with chloroquine, using the in vitro microtechnique. The growth in the control wells was between 43 and 200 schizonts per 200 asexual parasites, with a mean of 173 schizonts. Complete growth inhibition was observed as follows: Amount of No. ofcases chloroquine needed for complete growth inhibition (pmol) 2 2 4 8 5 9 6 6 8 4 12 2 The two samples that still showed schizont growth at 8 pmol were from patients with a parasitaemia of more than 100 000 parasites/ul of blood. Regression lines were therefore calculated separately for the groups with a parasitaemia of less or more than 100 000 parasites/;ul (Fig. 1). The regressions are significantly different. The best fit for the data from patients with parasitaemia below 100 000 parasites per ,AI was obtained with a logistic-log function using non- linear least squares analysis (Fig. 2). 100 90 80 H 70 60,, 60\ \ 50 a 40 5 30 20 10 0. 0.2 0.4 0.8 1.0 1.2 1.6 2.4 Chloroquine concentration (pmol/pl of blood) Fig. 1. P. falciparum schizont maturation determined by the in vitro microtechnique. Mean values and regression lines for blood samples containing < 105 (0) and > 105 (x) asexual parasites/,ul. SENSITIVITY OF P. FALCIPARUM IN SUDAN 0.2 0.4 0.8 1.0 1.2 Chloroquine concentration (pmol/pl of blood) Fig. 2. Non-linear least squares regression analysis of results of the in vitro microtest on 28 blood samples containing < 105 parasites/pi. Blood samples from 21 subjects were studied for the response of P.falciparum to Dabequin. Growth was totally inhibited at 1 pmol/well in 3 cases, at 2 pmol in 9 cases, and at 4 pmol in 20 cases. There was no growth at 5 pmol. The in vitro tests with mefloquine produced inconsistent results-a finding that has recently been shown to be associated with factors inherent in the preparation and the properties of the test plates. DISCUSSION The results of the in vivo studies showed no chloroquine resistance at RII and RIII levels. Similar observations by Omer (6) in the neighbouring Gezira irrigated area indicate that the P.falciparum strains prevailing in the area were sensitive to chloroquine. However, the mean clearance time of trophozoites from the blood was higher than in many other trials carried out in tropical Africa. It was also longer than those observed by Dennis et al. (7) and Armstrong et al. (8) in Ethiopian subjects with relatively low immunity, where the in vitro response of P.falci- parum approached the level of drug refractoriness. The in vitro results obtained with the micro- technique suggest that strains of P.falciparum found in the area of Sennar are less susceptible to chloro- quine than those found in other African countries. Four samples with a parasite density of less than 100 OOO/M1 of blood showed schizont maturation at a level of 6 pmol chloroquine per well (1.20 pmol per IAI of blood). This is close to the discriminatory level of 1.25 pmol per 1l of blood used in the standard in vitro macro test. Fig. 3 shows that the isolates from Sennar are approximately half as sensitive to chloroquine as the highly susceptible Uganda FUP strain, and over six times more sensitive than the FVO Viet Nam Oak Knoll strain and the Prabuddhabat (Thailand) iso- lates. This response is similar to that of Ethiopian strains observed by Dennis et al. (7) and Palmer et al. (9), who concluded that the relatively low sensitivity of these strains was a result of innate strain variation rather than strain selection under drug pressure. The significant difference between the regression lines for growth under chloroquine of blood samples containing less than, and those containing more than 100 000 parasites/ AI of blood can be explained by the selective uptake of chloroquine by the parasitized 90 80 Iu/U 0 -i 60 '.4 -. 50 co " 40 0 N *,1 i5 30 cn 20 10 0 787 R. L. KOUZNETSOV ET AL. 100 90 , 80 * 70 . __ 60 *\%4 40 * 30 * \ 20. *\ FUP* 10 *SNA 50 % .4h . _FV0 *~~~~~~~ _ * N 1 PRABUDDHABAT 1- _ _ 0.4 0.8 1.2 1.6 2.4 3.2 4.8 6.4 Chloroquine concentration (pmol/pl of blood) Fig. 3. Chloroquine sensitivity of P. falciparum determined by the in vitro microtest. FUP - Palo Alto, Uganda strain; FVO- Oak Knoll, Viet Nam strain; Prabuddhabat- mean of 8 isolates from Prabuddhabat, Thailand; Sennar- mean of 28 isolates from Sennar, Sudan. cells, necessitating higher levels of drug in samples with a high parasite density. This correlation would have to be quantified for the interpretation of tests using such material. The clinical response of patients with parasitaemia exceeding 100 000/Ml of blood was as quick and complete as that of patients with lower levels of parasitaemia. The results obtained with Dabequin using the microtechnique suggest that it has a significantly higher in vitro activity than chloroquine. In vivo studies are needed to compare the clinical activity of both compounds. In these studies, the modified in vitro micro- technique proved to be quite suitable for field use and we think, should prove to be more useful than the standard in vitro macromethod. ACKNOWLEDGEMENTS We are grateful to the staff of the Division of Malaria Control, Ministry of Health, Democratic Republic of the Sudan; to the personnel of the Malaria Training Centre, Hospital and Health Centres of Sennar; Professor A. H. S. Omer, University of Khartoum and Dr H. H. Mashaal, WHO Senior Malariologist. These studies were supported, in part, by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. 0 JJ 4-W 0 0, U4 788 SENSITIVITY OF P. FALCIPARUM IN SUDAN 789 RtSUME EMPLOI DE LA MICRO-TECHNIQUE IN VITRO POUR EVALUER LA SENSIBILITE AUX MEDICAMENTS DE PLASMODIUMFALCIPARUMA SENNAR (SOUDAN) L'&ude effectuee A Sennar (Soudan) avait pour objet 1) de definir la sensibilite des souches locales de Plasmodium falciparum A la chloroquine en utilisant aussi bien l'epreuve in vivo que la methode habituelle et la micro-technique in vitro; 2) d'obtenir des donnees de base sur la sensibilite de P.falciparum A de nouveaux medicaments comme la meflo- quine et la Dabequine (aminobenzo-4 quinoleines) en utili- sant la micro-technique in vitro; et 3) d'evaluer la possibilite d'une utilisation etendue de la micro-technique sur le terrain. Cette etude a e menee dans une region autrefois mesoendemique oil, apres 17 ans de lutte intensive, la trans- mission du paludisme avait &W largement enrayee et ou il est par consequent A craindre que l'immunite aussi ait e fortement abaiss&e. Les enquetes in vivo menees au moyen de l'epreuve type OMS ont indiqu6 une absence de resistance A la chloroquine aux niveaux RII et RIII mais n'ont pas exclu qu'elle soit possible au niveau RI. Neanmoins, il semblerait que les souches de P.falciparum que l'on rencontre dans la region soient sensibles A la chloroquine et que celles qui y sont resistantes soient tres peu nombreuses ou meme inexistantes. Dans la presente etude, toutes les tentatives d'evaluation de la sensibilite de P.falciparum A la chloroquine au moyen de la micro-technique in vitro ont e vaines, probablement A cause des difficultes rencontrees pour maintenir la tempera- ture d'incubation aussi pres que possible de 38,5C. L'absence de resultats obtenus A partir de la macro-tech- nique a complique l'interpretation des resultats de la micro- technique in vitro. Cependant, les resultats des etudes in vitro concordaient generalement avec ceux des observations in vivo et tendaient A confirmer que la reaction relativement lente, in vivo, de P.fakciparum A un traitement normal par la chloroquine dans cette region s'expliquait par la plus faible sensibilite du parasite en question A la chloroquine par rapport A d'autres souches africaines de P.falciparum, hormis peut-etre les souches ethiopiennes. L'utilisation des microplaques additionnees de meflo- quine A I'avance a donne des resultats contradictoires et, comme il semble que la matiere plastique des plaques retienne la mefloquine, de nouvelles 6tudes sont en cours pour mettre au point des plaques entierement bioactives destin&es A l'essai de la mefloquine. D'apres les resultats obtenus par micro-technique, la Dabequine, recemment mise au point en URSS, aurait une action nettement plus efficace que la chloroquine. Seules des etudes in vivo permettront de comparer l'action clinique de la Dabequine A celle de la chloroquine. Enfin, la pr6sente etude a confirme les enormes possi- bilites de la micro-technique in vitro. La contamination ne pose presque aucun probleme et on a decouvert que la genta- mycine est probablement le meilleur agent non parasiticide propre A empecher la croissance bacterienne dans les cultures A court terme. REFERENCES 1. WERNSDORFER, G. & WERNSDORFER, W. Malaria in mittleren Nilbecken und dessen Randgebieten. Zeitschrift far Tropenmedizin und Parasitologie, 18: 17-44 (1967). 2. WHO Technical Report Series, No. 529, 1973 (Chemo- therapy of malaria and resistance to antimalarials: report of a WHO Scientific Group). 3. RIECKMANN, K. H. ET AL. Drug sensitivity of Plasmodium falciparum. An in vitro microtechnique. Lancet, i: 22-23 (1978). 4. TRAGER, W. & JENSEN, J. B. Human malaria parasites in continuous culture. Science, 193: 673-675 (1976). 5. BRUCE-CHWATT, L. J. 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