World Health Organization ctd/mal/97. 20 Rev. 2 2001 Organisation Mondiale de la Santé distr.: Limited original: english
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H Test Number Control 1 pmol 2 pmol 4 pmol 8 pmol 16 pmol 32 pmol 64 pmol 1 190
164
81
43
11
2
0
0 2 112
64
32
0
0
0 0
0
178
179
160
138
111
67
34
0
4 196
192
190
186
110
65
23
0
5 49
53
21
11
2
0
0 0
6 34
32
30
21
6
1
0 0
7 200
191
111
65
38
11
3
0
29
13
3
0
0 0
0
0 9 128
64
30
16
2
0
0 0
10 55
53
47
26
12
0
0
0 As Percentages of Control:
1 100.0
86.3
42.6
22.6
5.8
1.1
0.0
0.0
2 100.0
57.1
28.6
0.0
0.0
0.0
0.0
0.0
3 100.0
100.6
89.9
77.5
62.4
37.6
19.1
0.0
4 100.0
98.0
96.9
94.9
56.1
33.2
11.7
0.0
5 100.01
08.2
42.9
22.5
1.0
0.0
0.0
0.0
6 100.0
94.1
88.2
61.8
17.6
2.9
0.0
0.0
7 100.0
95.5
55.5
32.5
19.0
5.5
1.5
0.0
8 100.0
44.8
10.3
0.0
0.0
0.0
0.0
0.0
9 100.0
50.0
23.4
12.5
1.6
0.0
0.0
0.0
10 100.0
96.4
85.5
47.3
21.6
0.0
0.0
0.0
TOTAL 1000.0
831.0
563.8
371.6
185.3
80.3
32.3
0.0
maturation (mean) 100.0
83.10
56.38
37.16
18.53
8.03
3.23
0.00
% inhibition (0)
16.90
43.62
62.84
81.47
91.97
96.77
10.0
CTD/MAL/97.20 Page 18
In the above example, there is obviously still significant schizont maturation in the wells containing 8 and 16 pmol chloroquine, and three out of ten isolates show schizont maturation even at 32 pmol. As indicated in the first table of this section, schizont maturation in the 8 pmol well is a sign of resistance. Eight of the ten isolates showed schizont maturation at this well. This series reflects a status of manifest resistance as also evident from the EC-data in Annex I.
The principal advantage of this system is the comparability of quantitative results, both between different geographical areas and between series from the same area at different times (longitudinal studies).
The thick film counts will produce data as shown in the following tabulated example:
Well
A B C D E F G H
Schizonts with 8 or more normal nuclei
55
56
47
8
0
0 0
0
Other schizonts and rings
145 144
153
192
200
200
200
200
% schizont maturation (% of control)
100
85
15
0
0
0 0
% inhibition of schizont maturation
0
15 85
100
100
100
100
NOTE: Well A = control; Well B = lowest concentration of SDX/PYR; Well H = highest concentration of SDX/PYR
The >breakpoint= in this example is therefore between well D (SDX 100 pmol / PYR 1.25 pmol per well) and well E (SDX 300 pmol / PYR 3.75 pmol per well). Data so far available indicate that the 90% inhibition level, related to schizonts of eight or more normal nuclei, reflects more accurately the true >breakpoint=, which can be readily ascertained by means of a simple diagram as shown in the figure, using the data given above.
Procedure and interpretation: The observed data are entered according to drug concentration (x-axis) and inhibition (y-axis). A line of the best optical fit is drawn. Reading this line off the vertical scale at 90% inhibition yields the approximate IC-90. Here the IC-90 for the combination of SDX/PYR shows approx. 110 pmol SDX and 1.4 pmol PYR per well. This corresponds to concentrations of 2.2 µmol SDX and 0.028 µmol PYR per litre blood-medium mixture (BMM)
10 100 1000
10000 0.1
1 10 0.125 1.25 12.5
125 pmol SDX
pmol PYR 90 CTD/MAL/97.20 ANNEX I Page 19
ANNEX I
CTD/MAL/97.20 ANNEX I Page 20
Study Area: Sampelia Year: 1997 Drug: Cholorquine
B C D E F G H Control SCHIZ Conc. SCHIZ Conc. SCHIZ Conc. SCHIZ Conc. SCHIZ Conc. SCHIZ Conc. SCHIZ Conc. SCHIZ 0
0.2
0.4 0.8
1.6
3.2
6.4
12.8
190
164
81
43
11
2
0
0 112
64
32
0
0
0 0
0
178 179
160
138
111
67
34
0
196
192 190
186
110
65
23
0
49
53 21
11
2
0
0 0
34
32 30
21
6
1
0 0
200
191 111
65
38
11
3
0
29 13
3
0
0 0
0
0 128
64
30
16
2
0
0 0
55
53 47
26
12
0
0
0
Drug Concentration SMI% 0.2
16.90
0.4
43.61
0.8
62.85
1.6
81.16
3.2
91.97
6.4
96.77
12.8
100.00
CTD/MAL/97.20 ANNEX I Page 21
n = 10
S =
3.3797
f s
1.7017 a =
5.4749
A =
1.4803
f EC-50
1.7047 b =
0.8166
K =
7
f EC-90
2.3940 r =
0.9949
N = = 40
f EC-95
2.7915 x 2
0.1992
R =
64
f EC-99
3.8898
95% Confidence Levels
Mean
Higher EC 1
0.0324 0.0083
0.1260
EC 16
0.1654 0.0970
0.2820
EC 50
0.5590 0.3279
0.9529
EC 84
1.8893 1.1083
3.2206
EC 90
2.6852 1.1217
6.4285
EC 95
4.1898 1.5009
11.6958
EC 99
9.6508 2.4810
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