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Field evaluation of the use of an ELISA to detect chloroquine and its metabolites in blood, urine and breast-milk
521
TKANS.WXIONSOF THE ROYAL SOCIETYOF TROPICALMEDICINEAND HYGIENE (1990) 84, 521-525
Field evaluation of the use of an ELISA to detect chloroquine metabolites in blood, urine and breast-milk
and its
A. M. C. Witte’, H. J. H. Klever’, B. J. Brabin2p4, T. A. Eggeite3, H. J. Van der Kaay’ and M. P. Alpers’ ‘Laboratoy of Parasitology, Institute of Tropical Medicine, University of Leiden, Post Box 9605,230O RC Leiden, The Netherlands; 2Papua New Guinea Institute of Medical Research, P.O. Box 378, Madang, Papua New Guinea; 3Royal Tropical Insitute, N. H. Swellengrebel Laboratory for Tropical Hygiene, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands; 4Department of Tropical Paediatrics and International Child Health, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 SQA, UK
Abstract The evaluation of an enzyme-linked immunosorbent assav (ELISA) for chloroauine and its metabolites in b&d, urine and breast-milk is reported. ELISA blood levels, following standard treatment with chloroquine of pregnant and non-pregnant women, showed mean values comparable to other analytical methods. Blood chloroquine concentrations were estimated at day 0, 35tiOO nglml; day 2, 1000-1500 ne/ml: dav 14. 350-400 r&ml; dav 28, 180-350 ng/&l. In a
~~
~~~
~~~~
to distinguish between insufficient dosage and true resistance, and to ensure that no additional chloroquine has been taken. An enzyme-linked immunosorbent assay (ELISA) for chloroquine and its metabolites has been developed by Eggelte (unpublished work) which is both sensitive and specific, does not cross-reactwith other antimalarial compounds, and can be readily adapted for use under field conditions. In this test specific anti-chloroquine antibodies are used which are directed against the 4-amino-7-chloroquinoline moiety of the chloroquine molecule. A recent field trial in The Gambia showed that this ELISA nerformed better than the Dill-Glazko or Haskins calorimetric tests (SHENTON et al., 1988). In comparison to the Dill-Glazko test. the ELISA is 100-1000 times more sensitive (5 ng/rnl can be detected), and this permits its use for assay of chloroquine in blood. 100 ~1 of finger-prick blood is sufficient for this test. The assay is semi-quantitative when results of several test sample dilutions are compared. In order to evaluate this ELISA under field conditions the present study had the following objectives. (i) Monitoring chloroquine and desethylchloroquine concentrations in blood, urine and breast-milk of women with I’. falciparum infections receiving standard treatment with chloroquine. (ii) Comparison of ELISA estimates of chloroquine in blood and urine with the results of the Dill-Glazko test on urine and with the chloroquine history of subjects. (iii) Monitoring comnliance with weeklv chldroqume chemoprophilaxis in pregnant women: This paper reports the results of the first 2 study objectives. Details of the chemoprophylaxis trial in pregnant women have been reported (BRABIN et al., 1990). Materials and Methods Study area and subjects
The study was -undertaken between November 1986and March 1987through the Alexishafen Health Centre, Madang Province,-Panua New Guinea. 48 pregnant and non-pregnant women were selected for inclusion in the studv who were on the World Health Organization (WH6) extended 28-day in vivo field test for susceptibility of P. falciparum to chloroquine. 9 of these women were studied from the time of delivery. A small number of children (mean age 12 years) attending the clinic at Alexishafen were also screenedto provide a broader range of samples from different agesin order to compare the ELISA blood
522
and urine values with history of chloroquine use in women and children. The study was approved by the National Medical Research Advisory Committee and the Tropical Disease Research Programme (WHO) Review Committee. All subjects gave verbal informed consent.
ponded to this cut-off between positive and negative samples. This sameinhibition value was used as the cut-off between positive and negative for urine and breast-milk samples.A positive result at 1:10 dilution equals approximately 50 ng chloroquine per ml; at 1:50 dilution, about 250 ng chloroquine per ml; and at 1:250 dilution, at least 1250 ng per ml.
Bleeding and field procedure
From each subject a chloroquine history was taken and a blood smear obtained for examination for malarial parasites. Those with malaria were treated with 25 mg chloroquine baseper kg over 3 d (10, 10, 5 mg), and they were observed to swallow their tablets. Subjects were requested to provide urine, blood (firmer-nrick) and. when relevant. breast-milk samples0; days 0, ‘1,2,4,7, 10, 14,21 and 28 of the follow-up period. 75 ul blood obtained in a heparinized capillary tube were expelled immediately in a 1:lO dilution of phosphate-buffered saline (PBS) containing 0.05% Tween-20. Lysis of red blood cells was induced by freezing and thawing. Urine and breast-milk samples were stored undiluted without a preservative within 3 h of collection at -10°C until the ELISA or Dill-Glazko test was performed.
Chloroquine ELISA
The ELISA for chloroquine was developed on the basis of monoclonal antibodies raised against a chloroquine-protein conjugate obtained from coupling hydroxychloroquine to bovine serum albumin. This monoclonal antibody recognizes the 4-amino-7chloroquinoline moiety of the chloroquine molecule and, therefore, also recognizes chloroquine metabolites and amodiaquine. To each well of a 96-well polystyrene microtitre plate (Greiner) were added 100 pl of the coating antigen (see below), 5 @ml in 0.015 M carbonate buffer (pH 9.6). Plates were incubated for 12 h at 37°C and washed with PBS/O-OS% Tween-20. Coating antigen was prepared by coupling 4-(l$diaminopropyl)-7-chloroquinoline to an activated polyacrylamide polymer. 50 ul of the blood, urine or breastmilk sampleswere added in the following dilutions in PBS/Tween-20: blood and breast-milk, l:lO, 1:50, 1:250; urine, l:lO, l:lOO, l:lOOO, 1:lO 000. Subsequently 50 ul of a dilution of a peroxidase-labelled antichloroquine antibody (F73-Cl-l; WILSON & NAKANE, 1978)were added. After mixing, the microtitre plate was incubated for 1 h at 37°C. Plates were then washed and, to eachwell, 100 ul of enzyme were added and the plate was examined after 1 h using a microtitre plate reader (Titertekm Flow Laboratories, Finland) at 492 nm. A calibration curve was made for each plate using standards of chloroquine diphosphate dissolved in distilled water. Results were expressedas percentage inhibition in ELISA optical densitv (OD) readings. The positivity cut-off was determined -from the mean inhibition value dus the 95% confidence interval for the 1:50 dilutionbf blood samplescollected on day 14 of the follow-up. This interval was chosen firstly to compare the ELISA results with the chloroquine history in the previous 2 weeks, and secondly to determine a specific blood dilution and absorbance value that would divide negative from positive at a standard period following therapeutic drug ingestion. An inhibition value of approximately 40% corres-
Dill-Glazko
test
50 mg of eosin were added to 100 ml of chloroform and 1 ml of hydrochloric acid. After shaking, the chloroform became light yellow in colour. The chloroform layer was separated and stored in a dry brown glass-stopperedbottle. 10 drops of this chloroform solution were added to 2 ml of urine in a test tube. After mixing, the presenceof chloroquine was indicated by a change in the colour of the precipitated layer from light yellow to violet-red (LELYVELD & KORTMANN, 1970). Statistical analysis
The x2 test with Yates correction was used for statistical analysis. Results Loneitudinal ELISA mi&
in whole blood. urine and breast-
Mean ELISA matrix values and 95% confidence intervals following chloroquine treatment of 48 women are shown in Fig. 1. For a few subjects samples were not available on certain days due to absenceat the time of the field visit. If a woman lived in a remote area day 4 and day 10 follow-ups were omitted. Using the calibration curve the mean blood chloroquine concentrations per ml can be estimated (from I:10 and 1:50 dilutions) as follows: dav 0. 350-400 rig/ml; day 2 1000-1500 &ml; day* 14; 350400 rig/ml; day 28, 180-350 rig/ml. Peak blood levels were reached at day 4 (at 1:10 dilution equivalent to 1.6 ug per ml), after which values declined. There was evidence that additional chloroquine was taken during follow-up by 2 women. Mean values for women studied in the ante-natal period or post-partum period were comparable. Pre-treatment values indicated chloroquine usage before treatment in many subjects. so
Fig. 1. MeanELISA values(andlower95%confidenceinterval)at three blood dilutions for samplesfrom 48 femalesubjectstaken beforeand aftertreatmentwith 25 mg chloroquinebaseper kg body weight.
523 Table 3. Sensitivity and positive predictive value of ELISA test on urine (1:lOOO)compared to ELBA test on blood
90 80 70 * 0
60
2 Y
50
0 0
4
16
12
8
1 20
------24
Fig. 2. Mean ELISA values (and lower 95% confidence interval) for breast-milk samples from 9 post-partum subjects followed from rhe time of delivery after treatment with 25 mg chloroquine base per kg body weight.
4
8
12
16
20
24
28
D&IS
Fig. 3. Mean ELISA values (and lower 95% confidence interval) for urine samples from 7 post-partum subjects followed from the rime of delivery after treatment with 25 mg chlorcquine base per kg body weight.
Table 1. Pi-Glazko positivity compared to ELBA positivity for urine samples from 7 women collected following standard treatment with chloroquine Follow-up day Number of samples Dill-Glazko positive (%) ELISA positive (%1
12 67 83 86 83 86
4 7 86 100
7 2 100 100
ELISA Blood dilution (n=77) 1:lO 1:50 1:250
Sensitivity (%) Specificity (%) Positive predictive value (%)
58 75 95
67 56 57
:z 14
28
OAYS
0
ELISA Urine dilution 1:lOOO (n=77)
10 14 21 28 3 4 6 6 67 50 0 0 100 75 50 0
Table 4. Comparison between the history of chloroquine use in the previous hvo weeks aad ELISA positivity at different blood dilutions for 190 subjects ELISA Dilution
Result
Chloroquine history Yes No
Y’
Significance ‘P)
1:lO
+
91 9
69 21
6.2
1:so
.c
66 34
31 ‘9
17.6
0~0001
1:250
4. .-
22 78
4 86
10.9
O~Olll
0.01
Nine subjectswere selectedfor study of breast-milk chloroquine concentrations following delivery. Breast-milk was not obtained on day 0 for 5 of these subjects becauseproduction was not established. For 3 women sampleswere collected only for the first 7 d post-partum. The mean values are shown in Fig. 2. Marginally higher mean concentrations were observed in breast-milk than in whole blood concentrations collected at the same follow-up period. Variation at day 10 may be explained by the low number of samples assayed at that time. ELISA values for urine tests on 7 post-partum subjects followed from the time of delivery are shown in Fig. 3. Difficulties were experienced in collecting urine samplesfrom women mostly becauseof cultural factors and inadequate toilet facilities under field conditions. Much higher concentrations of chloroquine in urine were observed throughout the followup period compared to whole blood or breast-milk values. At the 1:lOO dilution all urine samples remained strongly positive through the 28 d period. Comparison of chloroquine ELISA values with the Dill-Glazko test and history of chloroquine use
Table 2. Sensitivity, specificity and positive predictive value of Di-Glaako test on urine compared to ELISA test results on urine and blood
Dill-Glazko
Test
ELISA ELISA Blood dilution Urine dilution (n=77) (n=95) 1:10 1:lOO 1:lOOO 1:lOOOO 1:lO I:50 I:250 30 100
36 96
49 98
55 86
32 88
39 78
43 71
Positive predictive 100 value (%)
96
96
65
96
61
13
Sensitivity Specificity
(%) (%)
Table 1 compares Dill-Glazko values and ELISA positivity (at 1:lOOOdilution) in urine samples collected during the 28 d follow-up period. There was a declining percentage of urines giving a positive Dill-Glazko result in comparison to ELISA positivity. Day 28 showed negative results with both tests. Two samples from one woman collected on days 1 and 2 gave negative ELISA and Dill-Glazko tests. ELISA results with blood samples (I:50 dilution) collected from this patient were positive. It is probable that this person had not provided her own urine sample. The sensitivity and specificity of the Dill-Glazko test for ELISA urine and blood positivity are shown in Table 2. At higher urine dilutions (l:lOOO,
524
1:10 000) there was improved sensitivity and specificity, indicating better correlation with ELISA positivitv. The sensitivitv of the Dill-Glazko test did not exceed 55% at any urine dilution. There was a weak associationbetween results of the ELISA performed on urine and blood for 77 pairs of samples collected at the same time. The strongest associationwas between the 1:1000urine dilution &d the 150 blood dilution (x2=3.14, P=O*O76). Table 3 shows the sensitivity, specificity and positive predictive values of the ELISA test on urine at 1:lOOO dilution compared to the ELISA test on blood at 3 dilutions. A weak association was observed between ELISA results for breast-milk and blood for 55 paired samples collected at the same time. The highest x2 value was 0.151 for the 1:lO dilutions (P=O*697). Comparison between history of chloroquine use in the previous 2 weeks with results of the ELISA performed on blood showed significant correlations for all 3 blood dilutions (Table 4). The reliability of histories from adults and children was considered comparable, as childrens’ histories were provided by an accompanying adult. One-fifth of histories were for children. Discussion
The ELISA employing monoclonal antibodies for detecting chloroquine and its metabolites is a new and useful tool to study chloroquine levels in different body fluids, especially whole blood samples. An ELISA has been used by others for determining chloroquine in dried blood spots (ROWELLet al., 1988). ELISA blood levels following standard treatment with chloroquine show mean values broadly comparable to those reported in studies using high pressure liquid chromatography and ext&ti& fluorescence methods (WHITE. 1985). Since the ELISA measures both chloroquine and desethylchloroquine, nothing can be said about the ratio between these substances following treatment. ‘ROMBOet al. (1986) commented on the decreasing chloroquine/ desethylchloroquine ratio after chloroquine treatment, so that the contribution of the desethylchloroquine to the total antimalarial effect became more prominent. This is important becausedesethylchloroquine is less effective in vitro against chloroquineresistant strains (ADEROUNMU,1984; Fu et al., 1986). Assay methods which distinguish these 2 compounds would be preferable, particularly in the study of chloroquine-resistant -parasites. The cut-off for blood nositivitv chosen in this studv (inhibition value of 4i)%) is -comparable to that selected by SHENTONet al. (1988) ‘using the same ELISA techniaue for detecting chloroauine in urine. The cut-off in our study corre&onds t&a chloroquine concentration of about 5 ng per ml in an undiluted sample. Using this cut-off, the longitudinal values for ELISA positivity in urine (1: 1000 dilution) following standard treatment (Table 1) were comparable to those reported by SHENTONet al. (1988) following the standard treatment of children. The similarity of thesefindings in 2 very different populations indicates the suitability of this cut-off criterion for ELISA positivity. Selecting the cut-off for positivity at 40% inhibition was arbitrary but, as it related to the absorbance value of blood at 1:50 dilution 2 weeks after therapeutic drug ingestion, it indicated a suitable
ELISA value for potentially pharmacologically active levels of drug in the body. This cut-off value could be used to make decisions about subjects in drug sensitivity or pharmacokinetic studies in the field. Appreciable amounts of chloroquine were present in breast-milk, with mean ELISA values slightly higher than those for the corresponding whole blood samples. This may indicate that chloroquine is, to a small extent, concentrated in breast-milk. Breast-fed infants born to mothers on antenatal chloroquine chemoprophylaxis will receive small amounts of chloroauine in the first months of life. Others have also reported the presence of chloroquine in breastmilk (DETURMENYet al., 1984; OCUNBONAet al., 1987; EDSTEINet al., 1987). The effect of these low oral dosesof chloroquine in breast-fed infants on the acquisition ofinfant immunity to malaria or on the selection of resistant strains of P. fulcipurum in early infancy is unknown. The low correlation between ELISA positivity for blood and breast-milk may relate to differences in the exact time of collection of corresponding samples or differences between levels in colostrum and later breast-milk samples. This study confirms the low sensitivity and high specificity of the Dill-Glazko test (VERDIERet al., 1985; ROMBOet al., 1985; SHENTONet al., 1988). Contamination of urine with blood from menstruation could give a false positive result but is unlikely to have been of importance in these women as the majority were pregnant or post-partum. The Dill-Glazko test, although more simple to carry out than the ELISA, was 100-1000 times less sensitive. Under optimal conditions the ELISA could detect 10-20 ng chloroquine per ml of urine or whole blood or breast-milk, while the Dill-Glazko test detected only amounts greater than 5 ug/ml urine. Recent calorimetric tests on urine have greatly increased sensitivity compared to the Dill-Glazko (STEKETEEet al., 1988). A low correlation was observed between ELISA positivity in corresponding blood and urine samples. Blood samples were always collected before urine samples and time differences and problems in urine collection, as well as variations in diuresis, could explain the poor correlation. A 24 h urine collection would be preferable, but is difficult to achieve under field conditions as, even with spot urine collection in the present study, urine samples were difficult to obtain. This problem emphasizes the important advantage of the ELISA for chloroquine assays,as it provides direct information on blood concentrations. Other advantages include the suitability of fingerprick samples, especially in very young children, and these can concurrently be used for preparation of malaria slides. This is especially important for malaria prevalence surveys in which collection of large numbers of urine samples would be impractical. Prevalence figures could be readjusted to exclude malaria slide results for subjects with positive ELISA values. Further work needs to be done on the epidemiological applications of the ELISA for the interpretation of malaria prevalence data in areaswith high chloroquine utilization, and to study the efficacy of public health care in different social groups in connection with the use of antimalarial drugs. The positive pre-treatment ELISA values indicate significant chloroquine utilization in the study sample. The reliability of the chloroquine history in relation
525
to ELISA blood positivity was unexpected. Although statistically significant, the association was not very impressive since one-third or more subjects were positive in one and negative in the other. The validity of the history may be explained in part by the concurrence of a chloroquine chemoprophylaxis trial in pregnant women in this area. Women were familiar with the appearance of chloroquine tablets and a greater awarenessof their importance in pregnancy had been generated. Up to now there has been no established criterion for the concentration levels in blood which are acceptable for selection of patients for in vitro and in viva tests of drug sensitivity, or for assessmentof drug utilization. Information on chloroquine intake needed for selection of subjects can be obtained by using the ELISA on whole blood, and a suitable cut-off which relates to longitudinal values in blood has been proposed in this paper. Acknowledgements
We thank the following agencies for providingfunds to A. W. and H. K. for an elective period overseasto complete this project during their undergraduate medical course: Stichting Werkgroep Studiereizen Ontwikkelingslanden (WSO); Leids Universiteits Fonds; Papua New Guinea Institute of Medical Research.We thank Dr Karen Forsyth and her staff for technical assistancewith laboratory work; Meza Ginny for teaching two of us (A. W. and H. K.) Melanesian Pidgin and assistingwith follow-up; Miss J. Croon and J. Blotkamp for training in laboratory techniques; and Dr Peter Heywood and the staff of the Alexishafen Health Centre for their support for the project. We wish to acknowledge the UNDPWorld Bank/WHO Special Programme for Research and Training in Tropical Diseases (CHEMAL) and the Commission of European Communities ‘Medicine, Health and Nutrition in the Tropics’ for financial support. References
Aberounmu, A. F. (1984). In vitro assessment of the antimalarial activity of chloroquine and its major metabolites. Annals of Tropical Medicine and Parasitology, 78, 581-585. Brabin, B. J., Brabin, L., Ginny, M., Eggelte, T., Alpers, M. P. & Van der Kaay, H. J. (1990). Failure of chloroquine prophylaxis for falciparum malaria in pregnant women in Madang. Annals of Tropical Medicine and Parasitology, in press. Bruce-Chwatt, L. J., editor (1981). Chemotherapyof Malaria, 2nd edition. Geneva: World Health Organization. Deturmeny, E., Viala, A., Durand, A. & Nosny, Y. (1984). The occurrenceof chloroquine in milk, an account of one case. Therapie, 39, 428-440. Edstein, M., Veenendaal, R., Newman, K. & Hyslog, R. (1987). Excretion of chloroquine, dapsone and pyrimethamine in human milk. Britishyoumal of Clinical
Pharmacology, 22, 733-735.
Fu, S., Bjorkman, A., Wahlion,B., Ofori-Adjei, D., Ericsson, 0. & Sjoqvist, F. (1986). In tirro activity of chloroouine. the two enantiomers of chlorwuine. desethylchloro&re and pyronaridine against Piasmodium falcipamm. British Journal of Clinical Pharmacology, 22,
93-96. Klever, H. J. H., Witte, A. M. C., Brabin, B. J., Eggelte, T., Van der Kaav. H. 1. & Alters. M. P. 11988).In viva susceptibility of ~asn&arn falciphrum to chloroquine in pregnant and non-pregnant women and in vitro response of P. falciparum to chloroquine and mefloquine in Madang Province, Papua New Guinea. dcta Leidensia, 57, 29-38. Lelyveld, J. & Kortmann, H. (1970). The eosin colour test of Dill and Glaxko: a simple field test to detect chloroquine in urine. Bulletin of the World Health Organization,
42, 477-479.
Ogunbona, S. A., Onyeji, C. O., Bolaji, 0. 0. & Torimiro, S. E. A. (1987). Excretion of chloroquine and desethylchloroquine in human milk. British Journal of Clinical Pharmacology, 21, 473-476.
Rombo, L., Bjorkman, A., Sego, A., Lindstrom? B., Ericsson, 0. & Gustafsson, L. L. (1985). Reliabihty of the Dill-Glazko test. Lancet. i. 1509. Rombo, L., Bjorkman, A., Sego; A. & Ericsson, 0. (1986). Whole blood concentrations of chloroquine and desethylchloroquine during treatment of adult patients infected with Plasmodium vivax, P. ovale and P. malariae. Transactions of the Royal Society of Tropical Medicine and Hygiene, 80, 763-766.
Rowell, V., Rowell, F. J., Baher, A., Gavrie, D. & Sidhi, A. M. (1988). A specific ELISA method for determining chloroquine in urine or blood spots. Bulletin of the World Health Organization, 66, 211-217.
Shenton, F. C., Bots, M., Menon, A., Egglete, T. A., de Wit, M. &Greenwood, B. M. (1988). An ELISA test for detecting chloroquine in urine. Transactions of the Royal Society of Tropical Medicine and Hygiene, 82, 216-220. Steketee, R. W., Mount, D. L., Patcher, L. C., Williams. S. B., Churchill, F. C., Roberts, J. M., Kaseje, D. C. 0. & Brandling-Bennett, A. D. (1988). Field application of a calorimetric method of assaying chloroquine and desethylchloroquine in urine. Bulletin of the World Health Organization.
66. 485-490.
Verdi&, F., Rannun’amirija, J. A., Pussard, E., Clavier, F.. Biand? J. M. Coulanges, P. & Le Bras, J. (1985). Unrehability of the Dill-Glazko test in detecting chloroquine in urine. Lancet, i, 1282-1283. White, N. J. (1985). Clinical pharmacokinetics of antimalarial drugs. Clinical Pharmacokinetics, 10, 187-215. Wilson, M. B. & Nakane, P. G. (1978). Recent development in the periodate method of conjugating horse radish peroxidase (Z-IJG’) to antibodies. In: Immunofhwrescence and Related Staining Techniques, Knapp, W., Holdubar , R. & With, G. (editors). Amsterdam: Elsevier, North Holland, pp. 215-224. Received 23 March 1989; revised 29 September accepted for publication 1 February 1990
1989;
TKANS.WXIONSOF THE ROYAL SOCIETYOF TROPICALMEDICINEAND HYGIENE (1990) 84, 521-525
Field evaluation of the use of an ELISA to detect chloroquine metabolites in blood, urine and breast-milk
and its
A. M. C. Witte’, H. J. H. Klever’, B. J. Brabin2p4, T. A. Eggeite3, H. J. Van der Kaay’ and M. P. Alpers’ ‘Laboratoy of Parasitology, Institute of Tropical Medicine, University of Leiden, Post Box 9605,230O RC Leiden, The Netherlands; 2Papua New Guinea Institute of Medical Research, P.O. Box 378, Madang, Papua New Guinea; 3Royal Tropical Insitute, N. H. Swellengrebel Laboratory for Tropical Hygiene, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands; 4Department of Tropical Paediatrics and International Child Health, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 SQA, UK
Abstract The evaluation of an enzyme-linked immunosorbent assav (ELISA) for chloroauine and its metabolites in b&d, urine and breast-milk is reported. ELISA blood levels, following standard treatment with chloroquine of pregnant and non-pregnant women, showed mean values comparable to other analytical methods. Blood chloroquine concentrations were estimated at day 0, 35tiOO nglml; day 2, 1000-1500 ne/ml: dav 14. 350-400 r&ml; dav 28, 180-350 ng/&l. In a
~~
~~~
~~~~
to distinguish between insufficient dosage and true resistance, and to ensure that no additional chloroquine has been taken. An enzyme-linked immunosorbent assay (ELISA) for chloroquine and its metabolites has been developed by Eggelte (unpublished work) which is both sensitive and specific, does not cross-reactwith other antimalarial compounds, and can be readily adapted for use under field conditions. In this test specific anti-chloroquine antibodies are used which are directed against the 4-amino-7-chloroquinoline moiety of the chloroquine molecule. A recent field trial in The Gambia showed that this ELISA nerformed better than the Dill-Glazko or Haskins calorimetric tests (SHENTON et al., 1988). In comparison to the Dill-Glazko test. the ELISA is 100-1000 times more sensitive (5 ng/rnl can be detected), and this permits its use for assay of chloroquine in blood. 100 ~1 of finger-prick blood is sufficient for this test. The assay is semi-quantitative when results of several test sample dilutions are compared. In order to evaluate this ELISA under field conditions the present study had the following objectives. (i) Monitoring chloroquine and desethylchloroquine concentrations in blood, urine and breast-milk of women with I’. falciparum infections receiving standard treatment with chloroquine. (ii) Comparison of ELISA estimates of chloroquine in blood and urine with the results of the Dill-Glazko test on urine and with the chloroquine history of subjects. (iii) Monitoring comnliance with weeklv chldroqume chemoprophilaxis in pregnant women: This paper reports the results of the first 2 study objectives. Details of the chemoprophylaxis trial in pregnant women have been reported (BRABIN et al., 1990). Materials and Methods Study area and subjects
The study was -undertaken between November 1986and March 1987through the Alexishafen Health Centre, Madang Province,-Panua New Guinea. 48 pregnant and non-pregnant women were selected for inclusion in the studv who were on the World Health Organization (WH6) extended 28-day in vivo field test for susceptibility of P. falciparum to chloroquine. 9 of these women were studied from the time of delivery. A small number of children (mean age 12 years) attending the clinic at Alexishafen were also screenedto provide a broader range of samples from different agesin order to compare the ELISA blood
522
and urine values with history of chloroquine use in women and children. The study was approved by the National Medical Research Advisory Committee and the Tropical Disease Research Programme (WHO) Review Committee. All subjects gave verbal informed consent.
ponded to this cut-off between positive and negative samples. This sameinhibition value was used as the cut-off between positive and negative for urine and breast-milk samples.A positive result at 1:10 dilution equals approximately 50 ng chloroquine per ml; at 1:50 dilution, about 250 ng chloroquine per ml; and at 1:250 dilution, at least 1250 ng per ml.
Bleeding and field procedure
From each subject a chloroquine history was taken and a blood smear obtained for examination for malarial parasites. Those with malaria were treated with 25 mg chloroquine baseper kg over 3 d (10, 10, 5 mg), and they were observed to swallow their tablets. Subjects were requested to provide urine, blood (firmer-nrick) and. when relevant. breast-milk samples0; days 0, ‘1,2,4,7, 10, 14,21 and 28 of the follow-up period. 75 ul blood obtained in a heparinized capillary tube were expelled immediately in a 1:lO dilution of phosphate-buffered saline (PBS) containing 0.05% Tween-20. Lysis of red blood cells was induced by freezing and thawing. Urine and breast-milk samples were stored undiluted without a preservative within 3 h of collection at -10°C until the ELISA or Dill-Glazko test was performed.
Chloroquine ELISA
The ELISA for chloroquine was developed on the basis of monoclonal antibodies raised against a chloroquine-protein conjugate obtained from coupling hydroxychloroquine to bovine serum albumin. This monoclonal antibody recognizes the 4-amino-7chloroquinoline moiety of the chloroquine molecule and, therefore, also recognizes chloroquine metabolites and amodiaquine. To each well of a 96-well polystyrene microtitre plate (Greiner) were added 100 pl of the coating antigen (see below), 5 @ml in 0.015 M carbonate buffer (pH 9.6). Plates were incubated for 12 h at 37°C and washed with PBS/O-OS% Tween-20. Coating antigen was prepared by coupling 4-(l$diaminopropyl)-7-chloroquinoline to an activated polyacrylamide polymer. 50 ul of the blood, urine or breastmilk sampleswere added in the following dilutions in PBS/Tween-20: blood and breast-milk, l:lO, 1:50, 1:250; urine, l:lO, l:lOO, l:lOOO, 1:lO 000. Subsequently 50 ul of a dilution of a peroxidase-labelled antichloroquine antibody (F73-Cl-l; WILSON & NAKANE, 1978)were added. After mixing, the microtitre plate was incubated for 1 h at 37°C. Plates were then washed and, to eachwell, 100 ul of enzyme were added and the plate was examined after 1 h using a microtitre plate reader (Titertekm Flow Laboratories, Finland) at 492 nm. A calibration curve was made for each plate using standards of chloroquine diphosphate dissolved in distilled water. Results were expressedas percentage inhibition in ELISA optical densitv (OD) readings. The positivity cut-off was determined -from the mean inhibition value dus the 95% confidence interval for the 1:50 dilutionbf blood samplescollected on day 14 of the follow-up. This interval was chosen firstly to compare the ELISA results with the chloroquine history in the previous 2 weeks, and secondly to determine a specific blood dilution and absorbance value that would divide negative from positive at a standard period following therapeutic drug ingestion. An inhibition value of approximately 40% corres-
Dill-Glazko
test
50 mg of eosin were added to 100 ml of chloroform and 1 ml of hydrochloric acid. After shaking, the chloroform became light yellow in colour. The chloroform layer was separated and stored in a dry brown glass-stopperedbottle. 10 drops of this chloroform solution were added to 2 ml of urine in a test tube. After mixing, the presenceof chloroquine was indicated by a change in the colour of the precipitated layer from light yellow to violet-red (LELYVELD & KORTMANN, 1970). Statistical analysis
The x2 test with Yates correction was used for statistical analysis. Results Loneitudinal ELISA mi&
in whole blood. urine and breast-
Mean ELISA matrix values and 95% confidence intervals following chloroquine treatment of 48 women are shown in Fig. 1. For a few subjects samples were not available on certain days due to absenceat the time of the field visit. If a woman lived in a remote area day 4 and day 10 follow-ups were omitted. Using the calibration curve the mean blood chloroquine concentrations per ml can be estimated (from I:10 and 1:50 dilutions) as follows: dav 0. 350-400 rig/ml; day 2 1000-1500 &ml; day* 14; 350400 rig/ml; day 28, 180-350 rig/ml. Peak blood levels were reached at day 4 (at 1:10 dilution equivalent to 1.6 ug per ml), after which values declined. There was evidence that additional chloroquine was taken during follow-up by 2 women. Mean values for women studied in the ante-natal period or post-partum period were comparable. Pre-treatment values indicated chloroquine usage before treatment in many subjects. so
Fig. 1. MeanELISA values(andlower95%confidenceinterval)at three blood dilutions for samplesfrom 48 femalesubjectstaken beforeand aftertreatmentwith 25 mg chloroquinebaseper kg body weight.
523 Table 3. Sensitivity and positive predictive value of ELISA test on urine (1:lOOO)compared to ELBA test on blood
90 80 70 * 0
60
2 Y
50
0 0
4
16
12
8
1 20
------24
Fig. 2. Mean ELISA values (and lower 95% confidence interval) for breast-milk samples from 9 post-partum subjects followed from rhe time of delivery after treatment with 25 mg chloroquine base per kg body weight.
4
8
12
16
20
24
28
D&IS
Fig. 3. Mean ELISA values (and lower 95% confidence interval) for urine samples from 7 post-partum subjects followed from the rime of delivery after treatment with 25 mg chlorcquine base per kg body weight.
Table 1. Pi-Glazko positivity compared to ELBA positivity for urine samples from 7 women collected following standard treatment with chloroquine Follow-up day Number of samples Dill-Glazko positive (%) ELISA positive (%1
12 67 83 86 83 86
4 7 86 100
7 2 100 100
ELISA Blood dilution (n=77) 1:lO 1:50 1:250
Sensitivity (%) Specificity (%) Positive predictive value (%)
58 75 95
67 56 57
:z 14
28
OAYS
0
ELISA Urine dilution 1:lOOO (n=77)
10 14 21 28 3 4 6 6 67 50 0 0 100 75 50 0
Table 4. Comparison between the history of chloroquine use in the previous hvo weeks aad ELISA positivity at different blood dilutions for 190 subjects ELISA Dilution
Result
Chloroquine history Yes No
Y’
Significance ‘P)
1:lO
+
91 9
69 21
6.2
1:so
.c
66 34
31 ‘9
17.6
0~0001
1:250
4. .-
22 78
4 86
10.9
O~Olll
0.01
Nine subjectswere selectedfor study of breast-milk chloroquine concentrations following delivery. Breast-milk was not obtained on day 0 for 5 of these subjects becauseproduction was not established. For 3 women sampleswere collected only for the first 7 d post-partum. The mean values are shown in Fig. 2. Marginally higher mean concentrations were observed in breast-milk than in whole blood concentrations collected at the same follow-up period. Variation at day 10 may be explained by the low number of samples assayed at that time. ELISA values for urine tests on 7 post-partum subjects followed from the time of delivery are shown in Fig. 3. Difficulties were experienced in collecting urine samplesfrom women mostly becauseof cultural factors and inadequate toilet facilities under field conditions. Much higher concentrations of chloroquine in urine were observed throughout the followup period compared to whole blood or breast-milk values. At the 1:lOO dilution all urine samples remained strongly positive through the 28 d period. Comparison of chloroquine ELISA values with the Dill-Glazko test and history of chloroquine use
Table 2. Sensitivity, specificity and positive predictive value of Di-Glaako test on urine compared to ELISA test results on urine and blood
Dill-Glazko
Test
ELISA ELISA Blood dilution Urine dilution (n=77) (n=95) 1:10 1:lOO 1:lOOO 1:lOOOO 1:lO I:50 I:250 30 100
36 96
49 98
55 86
32 88
39 78
43 71
Positive predictive 100 value (%)
96
96
65
96
61
13
Sensitivity Specificity
(%) (%)
Table 1 compares Dill-Glazko values and ELISA positivity (at 1:lOOOdilution) in urine samples collected during the 28 d follow-up period. There was a declining percentage of urines giving a positive Dill-Glazko result in comparison to ELISA positivity. Day 28 showed negative results with both tests. Two samples from one woman collected on days 1 and 2 gave negative ELISA and Dill-Glazko tests. ELISA results with blood samples (I:50 dilution) collected from this patient were positive. It is probable that this person had not provided her own urine sample. The sensitivity and specificity of the Dill-Glazko test for ELISA urine and blood positivity are shown in Table 2. At higher urine dilutions (l:lOOO,
524
1:10 000) there was improved sensitivity and specificity, indicating better correlation with ELISA positivitv. The sensitivitv of the Dill-Glazko test did not exceed 55% at any urine dilution. There was a weak associationbetween results of the ELISA performed on urine and blood for 77 pairs of samples collected at the same time. The strongest associationwas between the 1:1000urine dilution &d the 150 blood dilution (x2=3.14, P=O*O76). Table 3 shows the sensitivity, specificity and positive predictive values of the ELISA test on urine at 1:lOOO dilution compared to the ELISA test on blood at 3 dilutions. A weak association was observed between ELISA results for breast-milk and blood for 55 paired samples collected at the same time. The highest x2 value was 0.151 for the 1:lO dilutions (P=O*697). Comparison between history of chloroquine use in the previous 2 weeks with results of the ELISA performed on blood showed significant correlations for all 3 blood dilutions (Table 4). The reliability of histories from adults and children was considered comparable, as childrens’ histories were provided by an accompanying adult. One-fifth of histories were for children. Discussion
The ELISA employing monoclonal antibodies for detecting chloroquine and its metabolites is a new and useful tool to study chloroquine levels in different body fluids, especially whole blood samples. An ELISA has been used by others for determining chloroquine in dried blood spots (ROWELLet al., 1988). ELISA blood levels following standard treatment with chloroquine show mean values broadly comparable to those reported in studies using high pressure liquid chromatography and ext&ti& fluorescence methods (WHITE. 1985). Since the ELISA measures both chloroquine and desethylchloroquine, nothing can be said about the ratio between these substances following treatment. ‘ROMBOet al. (1986) commented on the decreasing chloroquine/ desethylchloroquine ratio after chloroquine treatment, so that the contribution of the desethylchloroquine to the total antimalarial effect became more prominent. This is important becausedesethylchloroquine is less effective in vitro against chloroquineresistant strains (ADEROUNMU,1984; Fu et al., 1986). Assay methods which distinguish these 2 compounds would be preferable, particularly in the study of chloroquine-resistant -parasites. The cut-off for blood nositivitv chosen in this studv (inhibition value of 4i)%) is -comparable to that selected by SHENTONet al. (1988) ‘using the same ELISA techniaue for detecting chloroauine in urine. The cut-off in our study corre&onds t&a chloroquine concentration of about 5 ng per ml in an undiluted sample. Using this cut-off, the longitudinal values for ELISA positivity in urine (1: 1000 dilution) following standard treatment (Table 1) were comparable to those reported by SHENTONet al. (1988) following the standard treatment of children. The similarity of thesefindings in 2 very different populations indicates the suitability of this cut-off criterion for ELISA positivity. Selecting the cut-off for positivity at 40% inhibition was arbitrary but, as it related to the absorbance value of blood at 1:50 dilution 2 weeks after therapeutic drug ingestion, it indicated a suitable
ELISA value for potentially pharmacologically active levels of drug in the body. This cut-off value could be used to make decisions about subjects in drug sensitivity or pharmacokinetic studies in the field. Appreciable amounts of chloroquine were present in breast-milk, with mean ELISA values slightly higher than those for the corresponding whole blood samples. This may indicate that chloroquine is, to a small extent, concentrated in breast-milk. Breast-fed infants born to mothers on antenatal chloroquine chemoprophylaxis will receive small amounts of chloroauine in the first months of life. Others have also reported the presence of chloroquine in breastmilk (DETURMENYet al., 1984; OCUNBONAet al., 1987; EDSTEINet al., 1987). The effect of these low oral dosesof chloroquine in breast-fed infants on the acquisition ofinfant immunity to malaria or on the selection of resistant strains of P. fulcipurum in early infancy is unknown. The low correlation between ELISA positivity for blood and breast-milk may relate to differences in the exact time of collection of corresponding samples or differences between levels in colostrum and later breast-milk samples. This study confirms the low sensitivity and high specificity of the Dill-Glazko test (VERDIERet al., 1985; ROMBOet al., 1985; SHENTONet al., 1988). Contamination of urine with blood from menstruation could give a false positive result but is unlikely to have been of importance in these women as the majority were pregnant or post-partum. The Dill-Glazko test, although more simple to carry out than the ELISA, was 100-1000 times less sensitive. Under optimal conditions the ELISA could detect 10-20 ng chloroquine per ml of urine or whole blood or breast-milk, while the Dill-Glazko test detected only amounts greater than 5 ug/ml urine. Recent calorimetric tests on urine have greatly increased sensitivity compared to the Dill-Glazko (STEKETEEet al., 1988). A low correlation was observed between ELISA positivity in corresponding blood and urine samples. Blood samples were always collected before urine samples and time differences and problems in urine collection, as well as variations in diuresis, could explain the poor correlation. A 24 h urine collection would be preferable, but is difficult to achieve under field conditions as, even with spot urine collection in the present study, urine samples were difficult to obtain. This problem emphasizes the important advantage of the ELISA for chloroquine assays,as it provides direct information on blood concentrations. Other advantages include the suitability of fingerprick samples, especially in very young children, and these can concurrently be used for preparation of malaria slides. This is especially important for malaria prevalence surveys in which collection of large numbers of urine samples would be impractical. Prevalence figures could be readjusted to exclude malaria slide results for subjects with positive ELISA values. Further work needs to be done on the epidemiological applications of the ELISA for the interpretation of malaria prevalence data in areaswith high chloroquine utilization, and to study the efficacy of public health care in different social groups in connection with the use of antimalarial drugs. The positive pre-treatment ELISA values indicate significant chloroquine utilization in the study sample. The reliability of the chloroquine history in relation
525
to ELISA blood positivity was unexpected. Although statistically significant, the association was not very impressive since one-third or more subjects were positive in one and negative in the other. The validity of the history may be explained in part by the concurrence of a chloroquine chemoprophylaxis trial in pregnant women in this area. Women were familiar with the appearance of chloroquine tablets and a greater awarenessof their importance in pregnancy had been generated. Up to now there has been no established criterion for the concentration levels in blood which are acceptable for selection of patients for in vitro and in viva tests of drug sensitivity, or for assessmentof drug utilization. Information on chloroquine intake needed for selection of subjects can be obtained by using the ELISA on whole blood, and a suitable cut-off which relates to longitudinal values in blood has been proposed in this paper. Acknowledgements
We thank the following agencies for providingfunds to A. W. and H. K. for an elective period overseasto complete this project during their undergraduate medical course: Stichting Werkgroep Studiereizen Ontwikkelingslanden (WSO); Leids Universiteits Fonds; Papua New Guinea Institute of Medical Research.We thank Dr Karen Forsyth and her staff for technical assistancewith laboratory work; Meza Ginny for teaching two of us (A. W. and H. K.) Melanesian Pidgin and assistingwith follow-up; Miss J. Croon and J. Blotkamp for training in laboratory techniques; and Dr Peter Heywood and the staff of the Alexishafen Health Centre for their support for the project. We wish to acknowledge the UNDPWorld Bank/WHO Special Programme for Research and Training in Tropical Diseases (CHEMAL) and the Commission of European Communities ‘Medicine, Health and Nutrition in the Tropics’ for financial support. References
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Verdi&, F., Rannun’amirija, J. A., Pussard, E., Clavier, F.. Biand? J. M. Coulanges, P. & Le Bras, J. (1985). Unrehability of the Dill-Glazko test in detecting chloroquine in urine. Lancet, i, 1282-1283. White, N. J. (1985). Clinical pharmacokinetics of antimalarial drugs. Clinical Pharmacokinetics, 10, 187-215. Wilson, M. B. & Nakane, P. G. (1978). Recent development in the periodate method of conjugating horse radish peroxidase (Z-IJG’) to antibodies. In: Immunofhwrescence and Related Staining Techniques, Knapp, W., Holdubar , R. & With, G. (editors). Amsterdam: Elsevier, North Holland, pp. 215-224. Received 23 March 1989; revised 29 September accepted for publication 1 February 1990
1989;