The eosin colour test of Dill and Glazko: a simple field test to detect chloroquine in urine.

SMAIL SINGLE DOSES OF CHLOROQUINE AGAINST PLASMODIUM FALCIPARUM 477 the table in Pringle & Lane, 1966). It would thus appear that in order to obtain a probit for a TCD50, as a base-line against which possible changes in parasite susceptibility could later be measured, the test doses should vary between 1 mg/kg and 2 mg/kg of chloroquine. It should be stressed here that only the drug susceptibility of the infection can be measured, not that of the parasite. In a semi-immune population changes in this susceptibility may be caused by a variety of other factors in addition to sensitivity changes by the parasite strain. Decreasing immunity levels may be ofparamount importance in this respect. However, because the 5 mg/kg dose has been shown to produce consistently a 100% trophozoite clearance rate in asymptomatic carriers, double this dosage-i.e., 10 mg/kg-should be perfectly ade- quate for routine treatment of P. falciparum infec- tions occurring in the semi-immune population of north-eastern Tanzania. In the Mto wa Mbu settlement, where medicated salt (0.30% chloroquine) has been in use since 1961, all asymptomatic P. falciparum infections occurring in the local school population were proved to be fully susceptible to single-dose treatment with 2.5 mg/kg chloroquine. In the relatively small number of symptomatic P. falciparum infections that could be sufficiently followed up in the same area, all trophozoites disappeared within 4 days after administration of chloroquine in a single dose of 10 mg/kg. The long exposure to small chloroquine doses seems, therefore, not to have induced the appearance of less sensitive strains of P. falciparum in the area. ACKNOWLEDGEMENTS We thank Mrs Margaret Muniss, Mr Emanuel Elibariki, Mr George Kilua, Mr Theophili Mgheni and Mr B. Kilonzo for their assistance in these inves- tigations. We are especially grateful to Dr J. N. Raybould who generously gave his valuable time to assist us in the preparation of the manuscript. REFERENCES Bruce-Chwatt, L. J. (1968) Trans. roy. Soc. trop. Med. Hyg., 52, 389 Clyde, D. F. (1961) Amer. J. trop. Med. Hyg., 10, 1 Clyde, D. F. & Shute, G. T. (1957a) Centr. Afr. J. Med., 3, 496 Clyde, D. F. & Shute, G. T. (1957b) Trans. roy. Soc. trop. Med. Hyg., 51, 505 Jeffery, G. M. & Gibson, F. D. (1966) Bull. Wld Hlth Org., 35, 441 Laing, G. B. G. (1965) Preliminary observations on drug- resistance to malaria in northern Tanzania. In: East African Institute of Malaria and Vector-Borne Diseases, Annual report, 1963-64, Nairobi, pp. 5-9 Lelijveld, J. & Kortmann, H. (1970) Bull. Wld Hlth Org., 42, 477 Pringle, G. & Lane, F. C. T. (1966) E. Afr. med. J., 43, 575 WHO Scientific Group on Resistance of Malaria Parasites to Drugs (1965) Wld Hlth Org. techn. Rep. Ser., No. 296, pp. 33-39 Wolfe, H. L. & Hudlestone, J. A. (1969) Med. J. Zambia, 2, 173 The Eosin Colour Test of Dill and Glazko: a Simple Field Test to Detect Chloroquine in Urine by JAN LELIJVELD 1 & HENRI KORTMANN 2 In 1961, W. A. Dill and A. J. Glazko (personal communication) of Parke Davis & Company, Ann Arbor, Mich., USA, described a field test for the detection of amodiaquine in medicated salt. The method of preparation of the reagent used in this colour test, was as follows: 1 Director, East African Institute of Malaria and Vector- borne Diseases, Amani, Tanzania. 2 Medical Research Officer, East African Institute of Malaria and Vector-borne Diseases, Amani, Tanzania. " 50 mg of eosin (yellowish) are weighed and trans- ferred to a small glass-stoppered separatory funnel. 100 ml of chloroform (reagent grade) and 1 ml of IN hydrochloric acid are added, and the mixture is shaken by hand for a few minutes until the chloroform assumes a light-yellow colour due to solution of the eosin. The chloroform layer is allowed to separate, and may be transferred to a brown glass-stoppered bottle for storage." In the presence of an organic base, such as amodiaquine, the light-yellow colour of the un- 2488F NOTES FIG. I COMPARISON OF RESULTS OBTAINED WITH WILSON-EDESON TEST AND EOSIN COLOUR TEST FOR DETECTION OF CHLOROQUINE IN URINE SAMPLES FROM 20 PERSONS HAVING RECEIVED A SINGLE 10 mg/kg DOSE OF CHLOROQUINE 0 5 24 48 72 96 120 144 168 192 216 Hours after treatment EOSIN COLOUR TEST 0 5 24 481 72 96 120 144 168 192 216 Hours after treatment [520&{19--1-201 20 20119 12 191 20 No. of urines examined 119 120 20 2011gl219 20 19 I919 No. of urines examined FIG. 2 POSITIVE URINE SAMPLES IN EOSIN COLOUR TEST ACCORDING TO CHLOROQUINE DOSAGE WILSON- EDESON TEST 100 85 c # 58 ._s C2- 21 10 mg/kg 5 mg/ kg 2.5 mg/ kg 9k ~~~~~~~100 10096 81 8 71 63 ~~~~~~~~~~62 0-~~~~~~~~~~~~5 9 '0 24 48 72 96120 0 24 48 72 96 120 0 24 48 72. 96 120 Hours after treatment Hours after treatment Hours after treatment | 135 272 1 62 5 4 19119 18| 15 11 23 58 61 69 |5 57 No. of urines examined No. of urines examined No. of urines examined WHO 917b1 = x x § T t T | WW! q'-762 478 SIMPLE FIELD TEST TO DETECT CHLOROQUINE IN URINE 479 RESULTS OBTAINED BY THE WILSON-EDESON TEST AND THE EOSIN COLOUR TEST ON URINE SAMPLES COLLECTED 24 HOURS AFTER TREATMENT WITH EITHER CHLOROQUINE OR A PLACEBO No. Wilson-Edeson test Eosin colour test Treatment received of urines examined Positive Negative Inconclusive a Positive Negative Inconclusive a Chloroquine base(300 mg weekly) 114 99 13 2 100 14 0 Placebo (calcium lactate) 220 12 J 194 ] 14 ] 11 209 0 a These urines were not clear enough, even after filtration, to use the turbidity test. ionized eosin in chloroform is changed into a highly coloured and intensely fluorescent ionized form of eosin. Dill & Glazko mentioned that the eosin colour reaction could equally well be obtained with other organic bases such as chloroquine. Trials were therefore carried out to assess the efficacy of the test for detecting chloroquine in urine samples collected after treatment with the drug. Method Initially 20 employees of the East African Institute of Malaria and other Vector-Borne Diseases were given a single dose of 10 mg/kg of chloroquine.1 Urine samples were collected before treatment, 5 hours after treatment, and from then onwards at 24-hour intervals until all reactions became negative. These samples were tested for the presence of chloroquine with both the classical test of Wilson & Edeson (1954) and the Dill-Glazko eosin colour test. In the latter case the procedure was as follows. To a small test-tube containing about 2 ml of urine, 10 drops of the Dill-Glazko reagent were added and the contents mixed by vigorous shaking for a few moments. A colour change from yellowish to violet-red in the precipitated chloroform layer was taken as an indication of the presence of chloroquine in the urine. In the same way urine samples from a group of 334 pregnant women were routinely examined for the presence of chloroquine with both tests. These women were divided into 2 groups, 114 being given a weekly dose of 300 mg chloroquine for malaria prophylaxis and 220 receiving a tablet of calcium lactate as a placebo. Finally, the Dill-Glazko eosin colour test was used in the field to examine urines from groups of I Tablets of chloroquine diphosphate; all doses are given in terms of chloroquine base. persons who were given different single doses of chloroquine: 123 received a dose of 2.5 mg/kg; 70 a dose of 5.0 mg/kg and 135 a dose of 10 mg/kg. Results and interpretation The results of both tests are given in Fig. 1. They suggest that the Dill-Glazko eosin colour test is a suitable procedure for demonstrating the actual drug intake. However, results with this test are fully reliable only for a period of 48 hours after treatment. The Wilson-Edeson test on the other hand gave consistent results for a period of 96 hours after treatment. Nevertheless, the Dill-Glazko eosin colour test has the advantage of simplicity and a striking colour-change phenomenon, which distin- guishes immediately a positive from a negative reaction. The accompanying table presents results of tests carried out on pregnant women. The data confirm our observation that both tests appear to be equally sensitive at least during the first 24 hours after chloroquine administration. The eosin colour test, however, was shown to be reliable when used with urines that could not be made clear enough, even after filtration, to use the turbidity test of Wilson & Edeson. Fig. 2 shows results obtained in the field, from groups of persons who were given different single doses of chloroquine. These results indicate again that the test appears to be a reliable method to demonstrate actual drug intake. They indicate also the existence of a relationship between the dosage level employed and the period during which the drug can be demonstrated in the urine by this method. REFERENCES Wilson, T. & Edeson, J. F. D. (1954) Med. J. Malaya, 9, 115