NIed. & Biol. Eng. & Comput., 1978, 16, 408~-18
A multichannel ultrasonic activity monitor for in.vitro screening of antischistosomal drugs M . C. B r o w n Bioengineering and Medical Physics Unit, University of Liverpool, Liverpool L69 3BX, England
D. F. Norman
D. R, Bell
C . J . Chavasse
Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, England
A b s t r a c t - - A method of assessing the effect of antischistosomal drugs on the activity of schistosomes. in vitro has been devised and exploited. The system described records activity from the disturbances caused by the worm movements in a 10 MHz ultrasonic field, Activity recordings from up to 10 channels are plotted by computer, and changes in activity evaluated statistically. The activity changes in response to Metrifonate and to Oxamniquine are demonstrated and discussed
Keywords--Activity, Metrifonate, Oxamniquine, Schistosome, Ultrasonics
1 Introduction Bilharzia, is a parasitic disease of major medical and economic importance in the tropics. The symptoms are caused by large numbers of eggs laid by small worms (schistosomes) which live in the blood vessels of the gut or bladder. A major approach to the eradication of the disease is by chemotherapy to prevent the production of eggs, but the drugs which are currently available for this purpose are not ideal, either because of toxicity, lack of complete effectiveness, the need for multiple dosage, or cost. One possible way of evaluating antischistosomal drugs is to observe the effect they have on the activity of the worms maintained in vitro. The simple approach of visual observation has been used by COLES (1975) and other previous workers to obtain a qualitative idea of activity changes. Automated systems have been developed by HILLMAN and SE~qrx (1973) who detected the motion of the shadow cast by the worm, and by BROWN et al. (1973) using ultrasound. SCHISTOSOMIASIS, o r
2 Method The method employed here is a development of the ultrasonic method first described by BROWN et al. (1973). Each activity-monitoring unit uses a double ultrasonic transducer bonded onto the bottom of a glass vessel in which the schistosomes (approximately 10 mm long by 0" 5 m m diameter) are maintained in a flowing culture medium which can be modified by the addition or removal of the drug under test. The standing wave pattern of the ultrasound Received 12th August 1977
field inside the vessel is modified every time a worm, or part of a worm, moves, so causing a transient amplitude change in the sound level detected by the receiving transducer. Electrical signals corresponding to these amplitude disturbances are extracted by synchronous detection. F o r continuous worm movements, the signal from the detector is similar to that from Doppler velocity detection systems. The generation of activity signals may be clarified by reference to Fig. 1. The worm is considered here to be made up of small segments each reflecting the same ultrasonic energy and moving with a velocity component 11, away from the ultrasound transmitter/ receiver unit. The receiving ultrasonic transducer will detect a signal reflected from each segment which will have the form
where A is the reflected ultrasonic signal amplitude received from each segment, co is the ultrasonic angular frequency, c is the velocity of sound in the culture medium, 11, is the vertical velocity component of segment, t is the time, in seconds, and 0, is the phase angle of reflected signal from segment at t = 0. F o r the whole of the worm, segments 1 to N, the received signal will be 1N
F(t) = ~ A sin
[(
2V, c o ] ] co+ ~ / t + 0 , , /
V.+c]
j
This ultrasound signal, when amplified and detected synchronously by mixing with the transmitted
0140-0115/78/0755-0000 $1 950/0 9 IFMBE : 1978
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July 1978
signal, has low-frequency electrical components of the form
E(t) = ~ K , A s i n [ (2V,,co) t+(O.-Or)
L V.+c
W h e r e K x is the amplification of preamplifier and Or is the reference phase of transmit signal. In our system this signal E(t) is amplified and bandpass filtered to select a band of frequencies corresponding to a limited range of velocities. Thus, the activity signal can be represented.
N
(2V, o~)
. . . .
E'(t) = 2a/rx2A Sln 1
_ . ~ V.+c
t
where 0.01 < V, < 1.0 mm/s and K2 is the combined amlolication factor including bandpass filter. The particular velocity range was selected after signal and noise measurements on narrow frequency bands during a period of typical worm activity. The lower cut-off frequency is also required as a d.c. block. A figure for activity is derived from E'(t) by taking the mean modulus over a period less than the sampling intervals of the logging system. This is achieved by a rectifier and an analogue averager. This arrangement is seen on the block diagram in Fig. 2.
3 Apparatus A culture system is required to supply the worms with nutrients and with drugs. A continuous-flow system rather than an intermittent irrigation system was chosen because of the desire to leave the worms undisturbed except for the experimental agents. However, intermittent flow systems have been used
////
/ ///
VELOCITY
INCIDENT AND If' REFLECTED ULTRASOUND
by others, such as SENFTet al. (1973). The fluid-flow system employed here is that shown in diagram form in Fig. 3. Unsteady flow caused by pump peristaltic action, motor noise or movements of the tubes are interpreted by the apparatus as worm activity since the worms are displaced by sudden changes in flow rate. Disturbances in the flow are partially damped by the use of soft narrow-bore feed tubes. Also, the roller pump was chosen for its relatively smooth action. The rate of fluid flow is governed by the rate required for nutrition, the cost of the culture fluid and the time taken for the test drugs to reach full concentration at the worms. The culture vessel itself is a U-shaped Pyrex glass tube of 6 mm o.d. and 3 m m i.d., and the worms normally remain at the bottom of the U-bend, in the ultrasonic field. Fig. 4 shows a detail of this. The transducers are PZT4 material cemented onto a flattened section on the Pyrex U-tube and the piezoelectric element is scribed on the back to produce three receiving and three transmitting elements, as shown in Fig. 5, with the intention 0f obtaining sensitivity to movement over an adequate length of the culture vessel. The actual field pattern inside the vessels is, of course, impossible to standardise exactly, but bonding the transducer to the vessel removes the variability of temporary coupling mechanisms used with unbonded transducers, and reduces microphony due to microscopic motion between the transducer and the vessel. The 10 MHz drive signal to the transmit elements in the transducers is derived from a t.t.l, crystal oscillator and amplified by 10 independent drive amplifiers, each with controllable output. The receive elements are coupled to a l0 MHz tuned amplifier and diode detector. At the detector the breakthrough signal from the transmitter is so large
VECTORS
(Vr~)
Fig. 1 Simplified visualisation of the interaction o f elemental worm seEments with the ultrasound beam. The worm is represented as lying in the path o f a parallel ultrasonic beam and is in an aqueous culture medium
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409
The actual time constant used is important since the voltages existing at the output of the averagers are time weighted by worm activity occurring in the immediate preceding period. Also s o m e element of the previous voltage sample will be present in each value. This error in the signal must be kept small or removed in the computer program. The data recording system used is an autoranging lO-channel digital voltmeter which records the time, channel numbers, and voltage samples from the output points on the averagers onto magnetic cassette tape. When the averager time constants are set to 30s, the voltages are normally sampled at 1 rain intervals. The data has been transferred to punched paper tape before filing on the Liverpool University ICL 1906S computer and programs have been developed to convert the readings into a logarithmic form and to produce logarithmic plots. Also by selecting time blocks, histograms of the amplitudes of readings can be produced to compare different periods (blocks) during the experiments. Using these same time
that synchronous detection occurs and the output contains the motion information. The relevant frequency components are selected for amplification by a d.c. block and h.f. cut with 3 dB points at 0-16 Hz and 14Hz. All these sections are housed close to the transducers inside a heated room. The signals are then transferred to the main equipment console in an adjacent room and applied to a true rectifier circuit to provide a unidirectional signal for determining the modulus. The signals from this point in the processing are displayed on a small oscilloscope adjacent to the experiment, and also on a large display in the recording room. Fig. 6 shows some typical displayed signals for two channels, The average amplitudes of these signals are obtained by an analogue averager (r = 30s) to remove short-term changes and produce a d.c. voltage which represents the average amplitude of the activity signal during the previous 30 s. The time constant of the averager circuits can be changed to suit the rate at which activity is to be sampled by the logging system, CULTURE VESSEL
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Fig. 2 Block diagram of one channel of the monitoring system
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Medical & Biological Engineering & Computing
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blocks, mean and standard-deviation figures are produced for statistical consideration. 4 Results
Some of the earlier results have been used to fix the parameters of the present system. On recording activity by this method it is found that the sampled voltages may vary by two orders of magnitude (40 dB) over a period of 1-2 h. Variations between consecutive minute samples may be up to 20 dB although they are generally much less. When the worms are disturbed, activity may change by 40-80 dB in a very short time. Logarithmic presentation was therefore essential, but it has also proved to be appropriate to the behaviour patterns exhibited. In stable periods of
undisturbed activity the distribution of logarithmic values is near normal about the mean. Also, small induced changes in activity show good proportionality to the starting activity, suggesting that the basic activity pattern is logarithmic. The frequency spectrum of the Doppler signal (at the detector) was tested to find the correct bandpass characteristics for the amplifiers and Fig. 7 shows a result for a short period of recording. Maximum signal to noise ratio (and maximum signals) occur at very low Doppler frequencies, corresponding to worm movements which would be barely perceptible without a microscope. (A Doppler frequency of 1 Hz corresponds approximately to 0- 075 mm /s). We therefore decided that only frequencies below 14 Hz be included in the activity readings to restrict the noise bandwidth. It is important, however, when analysing the signal, to remember that no velocity information is included in the activity readings. 4.1 Sample experiments
Fig. 4 The culture vessel showing the bonded transducer and worm
Fig. 8a shows the computer activity plot recorded from two pairs of worms which were used as one of the controls for the experiment of Fig. 10. After approximately 2"5 h of recording, beginning immediately after removal from a mouse, the source of culture medium was changed (point X). After a further 2 h the worms were removed from the culture vessel (point Y) for microscopic examination, to establish a background noise level for the ultrasonic monitoring system. The activity plot is set out on a logarithmic scale based on l ' O m V output from the monitoring system and consists of samples recorded at 1 min intervals. Experimental and control graphs are
GLASS
Fig. 5 Section of transducer showing construction. The piezoelectric element is bonded on to a flat area ground on the base of the culture vessel. The piezoelectric element is 8 X 5 mm and the silvering on the back is scribed to make six narrow elements which are connected up as alternate transmit and receive transducers
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July 1978
411
normally presented superimposed, but are separated here for clarity. In this test the worms had a high activity when first removed from the mouse but became less active over the first hour. This is the usual behaviour.
Fig. 6 Activity signals seen on main display unit connected to the output of the true rectifier circuit. Sweep speed was 3 cm/s, and all signals are negative
After the first hour activity shows a relatively steady average activity with short-term variations of 6-10 dB. After the change to the second source of culture medium the mean activity looks slightly higher. This may be due to a very small difference between the two sources of culture media but is found to be statistically insignificant. After removal of the worms the background noise level is seen to be more than 30 dB below the previous mean activity level. To quantify these results, time blocks are selected during each phase of the experiment. Blocks are selected which have a steady mean activity, and
where possible after the transient effects on activity caused by interference with the apparatus or change in the culture medium have subsided. Here blocks AA and BB are the last hour before the next change in each case. The activity recordings within the selected time blocks are processed and presented as shown on Figs. 8b and 8c. A histogram of the frequency of activity samples falling within 2 dB bands gives a view of the level and spread of the samples. The calculated mean and standard deviation is also presented. The mean and standard deviation for the noise level is given in Fig. 8 caption. An indication of the real change in activity between blocks AA and BB can be gained from a simple comparison of the means. The difference in the means is 0"142 on the log scale. However, the application of a null hypothesis test (students t test) shows that p g 0" 05 suggesting that the activity may be unchanged. Fig. 9 shows an experiment of similar format but employing an organophosphorus compound (Metrifonate 10-4M) in the culture medium introduced at point X. Metrifonate is used to treat Schistosomiasis and is known to depress motor activity. The activity before the introduction of the drug did not stabilise and is therefore difficult to quantify. However, after point X there was a marked reduction in activity by approximately 60 dB. The output voltages after point X are near to the noise level of time block CC, and therefore represent minimal activity. The small difference between this suppressed
Fig. 7 Relative amplitude of the Doppler frequency components recorded from the detector using five worms, and then dead worms. The contribution from Doppler signals above 5 Hz is negligible. Also maximum signal to noise ratio of the overa# system (live worms against dead worms) occurs in the lowest frequency block
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Medical & Biological Engineering & Computing
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activity and the noise level could be due to the activity associated with the living state of the
paralysed worms but it is m o r e likely that their presence in the culture vessel acts as an extra noise
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Fig. 8 Record of activity of t w o pairs of worms freshly removed from a mouse and cultured in 50% human serum and 50% lactalbumin hydrolysate solution ( Gibco Bio-Cult, Paisley, Scotland--Cat. No. 125) until point X at which the culture medium was changed to a different source of the same fluid, as a control to experiments in which the new medium contained a drug.
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(a) Activity plot, using one minute sampling intervals and a logarithmic activity scale. Noise level is established with the worms removed at the end o f the test. The mean and standard deviations of the noise level are : M ---- O" 845, o" = 0 . 0 6 8 on the log scale using IV = 61 samples in block CC (b) and (c) The distribution of activity samples recorded in time blocks AA and BB, respectively, together with the mean and standard deviations J u l y 1978
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(a) Erratic but high activity of the fresh worms followed by an apparent total suppression of activity on introduction of the new culture medium containing the drug at point X. After a time the worms recovered 414
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6
(b) and (c) The large difference between the activities before and after the introduction of the drug. The large spread in readings is caused by the change in the mean activity occurring during the two time blocks selected for comparison. The noise level established in block CC was : M = 1.022, (7 = O" 063 on the log scale for N ~ 41 samples
M e d i c a l & Biological Engineering & C o m p u t i n g
J u l y 1978
source in response to background vibrations of the apparatus, After about 30 minutes at this low level the activity recovered slowly through time block BB. This recovery may be caused by the worms overcoming the chemical blocking action of the drug or by hydrolysis
~
3,5
of the drug in solution (ARTHUR and CASIDA, 1957). The histograms of Figs. 9b and 9c show non-normal distributions of readings, which is attributable to the non-stationary nature of the mean activity in both time blocks. A different drug, oxamniquine, was used in the
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Fig, 10Effect of Oxamniquine 7"16 x 1 0 - 4 M on one pa# of worms in a single channel (a) Erratic activity in the initial control phase is f o l l o w e d by a raised and very stable activity after introduction of the drug
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(b) and (c) demonstrate these changes. The noise /eve/ established from block CC was: M = 0"627, ~T ----- O" 069 on the log scale for N = 61 samples
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415
experiment presented in Fig. 10. The two pairs of worms showed continuous changes in activity throughout the first phase, and after introduction of the new culture medium containing oxamniquine 7 . 1 6 x 10-*M, the activity is seen to rise sharply. The comparison of results is more clear on Figs. 10b and 10c. The wide spread of results in block A A is due to the movement of the mean. A most interesting effect of the drug, in addition to the increase in activity, is that the range of readings is confined to a much narrower band. We have noticed this short term effect of oxamniquine on other occasions. The change in mean activity between blocks A A and BB was 0-6333 on the log scale (p < 0.01). 5 Discussion The method described here for evaluating antischistosomal drugs may be questioned in three ways. First, is the ultrasonic method appropriate ? Secondly, how should 'activity' be defined? Thirdly, can 'activity' be usefully related to the effectiveness of the drugs ? Clearly the ultrasonic method has the drawback that the worms are exposed to the ultrasonic waves, creating an environment which is abnormal and possibly damaging. Total electrical power levels delivered to each transducer is of the order of 20 m W and the ultrasonic power in the sonicated zone will be rather less. It is not possible to quantify the actual sound intensity reaching the worms because of the complex shape of the vessels and the standing wave pattern. The worms, which are constantly moving in the ultrasound field, are assumed to absorb only a small portion of the total power. Some experiments were conducted involving sonication for a period of a week, after which time most worms die due to some deficiency in their environment, or delayed effects of the mechanical damage during initial removal from the mouse. N o difference in life span has been found between the sonicated and unsonicated worms maintained in otherwise identical culture conditions. Also, sonicated and unsonicated worms examined by electron microscopy show no obvious differences. Although it seems likely that the ultrasound must affect the results in some way, the effects do not appear to present serious problems. The range of activity levels discernible by the ultrasonic method is large and on occasions may exceed the dynamic range capabilities of the recording system. However, the sensitivity to small movements seems excessive at times because background vibration, even vehicles passing the building, sometimes register as activity. The most irritating feature of the ultrasonic system is its great sensitivity to the bubbles which form in the culture medium by outgassing, or from small leaks. These cause very high noise levels that register as activity which can be applied to the motion of these small worms. COLES 416
(1975) classified the worms as active, slightly active or dead. HENRY et al. (1976) also using visual observations defined four levels of activity. The two moving shadow detectors of HILLMAN and SENF~ (1973) and that of JEWSBURY et al. (1977) derive values for activity in different ways. In the apparatus used by HILLMAN and SENFT the shadow cast by the worm moves over a matrix of 19 optical fibre terminations. Photocells at the other ends of the fibres register the number of times the shadow passes. One activity count is registered for each time that any fibre is shaded. Their activity plot therefore represents, in a complex way, an index of how much the worm has moved in a horizontal plane, without differentiating movements back and forth from continuous translation. Because of the 19 discrete sensors and the digital mode of processing it can be seen that small movements may not be registered. The method has, however, been used extensively, and appears to give useful and meaningful results (SENFI and HILLMAN, 1973; HILLMAN et al., 1974; HILLMANet al., 1975). The moving shadow method of JEWSBURY,et al. (1977) is really a simplification of the method of HILLMAN and SENFT. A single light-dependent resistor (l.d.r.) replaces the multiple-fibre detection system. The l.d.r, element is already in the form of a grid, and is a suitable size to fit underneath the culture vessel. An indian-ink grid scribed on clear perspex is placed between the worm and 1.d.r. so that as the worm moves its shadow falls on different parts of the matrix of sensing elements formed by the scribed grid and the l.d.r, element. At present, the method of analysing the resistance variations caused by the movement of the shadow is to count the number of turning points on a chart record of resistance. The figure should correspond to the number of matrix squares covered and uncovered during the period under examination. In spite of a rather subjective analysis and likely linearity defects of the sensing system it appears to work well, and it is attractive because of its simplicity. Activity as recorded by the ultrasonic method of BROWN et aL (1973) is the time average of the amplitude of the ultrasound reflected from those parts of the worm which are moving within an accepted velocity band. The amplitude of the reflected sound depends largely on the area projected by the worms on the base of the culture vessel (the number of elemental segments as in Fig. 1). Unevenness in the field pattern, standing waves and uneven sensitivity of the receiving elements are of relatively small importance, as the worms will normally move throughout the sensitive zone. This argument can also be applied to the alternative methods. The signal available at the detector in each channel contains velocity information in the form of Doppler frequencies. However, it was not thought worthwhile to use this information since the power spectrum of the Doppler signal (Fig. 7) shows that there is little
Medical & Biological Engineering & Computing
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signal energy associated with the motion occurring at visible velocities. The more rapid (and visible) movements occur relatively infrequently and involve less of the length of the worm. Calibration of the response of the measuring system and defining an index of activity has been a difficult problem with all methods. During experiments, there is a datum level created by the mean activity of the worms before the addition of the drug, and a base line is provided by the noise level of the apparatus with the worms dead or removed. The use of a logarithmic scale for presentation and analysis has the added advantage here that the gain of the sensing and amplifying system is not relevant provided adequate signal to noise ratio is achieved. A problem experienced with the simple form of analogue averaging employed here is that consecutive readings taken by the data recording system are not entirely independent--a small proportion of the previous reading remains due to the time constant of the averaging circuit. This effect has been minimised by using a sampling period of not less than twice the average time constant and also by developing a computer program to remove the residual portion of previous readings. Considering the third question, of whether activity is really relevant in evaluating the performance of autischistosomal drugs, is more difficult. Antischistosomal drugs may be intended to kill the worms directly, paralyse them so that they are carried away by the blood, prevent them from mating, or prevent egg laying. Not all of these approaches would necessarily result in gross activity modification. We have already seen that activity may decrease or increase in response to drugs which are known to be clinically effective, and fortunately currently used drugs do appear to modify in vitro activity at concentrations corresponding to those achieved in the treatment of human patients. Our recordings, and those of other workers, usually show a change in activity when the new agents are introduced which can be seen clearly on the graphs. However, interpreting these changes quantitatively is more difficult. A major problem is that mean activity is not normally the same for more than a short period, and, in some cases, particularly with single worms, the mean is continually changing over a wide range, perhaps with some periodicity. So it is difficult to capture a period of stationary activity, and often impossible to attribute a change in activity to the new agent with certainty, without resorting to many repeats of the experiment. F o r experiments of more than one day, stationary statistics may be inadequate. There is obviously other information about activity which could be derived, such as the magnitude of frequency shift and the patterns of activity variation. However, these could be explored in the future. Medical & Biological Engineering & Computing G
6 Conclusions
The ultrasonic method of activity measurement, the signal processing and data processing procedures presented here have been applied successfully to small worms (schistosomes). The method has some attractive theoretical features and produces high resolution plots of activity during experiments on the effect of drugs on the schistosomes. Changes in the level and in the pattern of activity can be demonstrated. By comparison with alternative (optical) methods it appears that this method introduces more practical problems, but its resolution and the possibilities for statistical analysis are good. Variations in individual worm activity have been found to be large compared to some of the effects under study. Appendix Manufacturers Ismatec MP25F Autoanalyser pump Ismatec S.A. Limmatstrasse 109 8031 Zurich Switzerland
Ultrasonic Transducers Transducer Manufacturing Services Lauriston Cowbridge Hill Malmesbury Wiltshire SN16 9UI UK Data Logging System P.C.D. Ltd. Alpha Chambers Alexandra Road Farnborough Road Hampshire GU14 6BU UK
Acknowledgments This
work was conducted with support from the Edna McConnell Clark Foundation (Grant No. 276-4}013). The authors would also like to express their gratitude to Prof H. M. Gilles for providing facilities at the Liverpool School of Tropical Medicine. The samples of Metrifonate were kindly donated by Bayer A. G. Wuppertal-Eberfeld (Pt Nr. 9298/75) and the Oxamniquine by Pfizer, Sandwich, Kent, UK (lot no. 272-02). References ARTHUR, B. W., CASIDA, J. E. (1957) Metabolism and selectivity of O, O-dimethyl 2,2,2-trichlorol-l-hydroxyethyl phosphonate and its acetyl and derivatives, jr. Ag. & Food Chem. 5, 186-192. July 1978
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BROWN, M. C., KOURA, M., BELL, D. R., GILLES, H. M. (1973) An in vitro activity monitor for schistosomes: preliminary report. Ann. Trop. Med. & Parasit. 67, 3, 369-370. COLES,G. C. (1975) The response of Schistosoma mansoni maintained in vitro to schistosomicidal compounds. J. Helminth. 49, 205-209. HENRY, W., GIRGIS, N. I., MANSOUR, N. S. (1976) In vitro studies on the use of penicillamine with tartar emetic against Schistosoma mansoni worms. Ann. Trop. Med. & Parasit. 70, 425-429. HILLMAN, G. R., OLSEN, N. J., SENFT, A. W. (1974) Effect on Methysergide and Dihydroergotamine on Schistosoma mansoni. J. Pharmacol. & Exp. Therapeutics 188, 529-535.
HILLMAN, G. R., SENFT, A. W. (1973) Schistosome motility measurements: Response to drugs, ibid. 185, 177-184. HILLMAN, G. R., SENFT, A. W. (1975) Anticholinergic properties of the antischistosomal drug Hycanthone. Am. J. Trop. Med. & Hygiene 24, 877-884. JEWSBURY,J. M., HOMEWOOD,C. A., MARSHALL,I. (1977) Inexpensive apparatus for measuring activity of Schistosoma mansoni in vitro. Trans. R. Soc. Trop. Med. & Hygiene 71, 115.
SENFT,A. W., HILLMAN,G. R. (1973) Effect of Hycanthone Niridazole and Antimony Tartrate on schistosome motility. Am. J. Trop. Med. & Hygiene 22, 734-742.
Controle d'activitd ultrasonore, a canaux multiples, pour tri prealable, in vitro, des medicaments anti-schistosomes Sommaire---I1 d6crit une m6thode de mise au point et d'application in vitro, de l'effet de m6dicaments antischistosome sur l'activit6 des schistosomes. Le syst6me d6crit enregistre l'activit6 de troubles caus6s par les mouvements des vers, dans un champ ultrasonore de 10 MHz. Les enregistrements de l'activit6, allant jusqu'& 10 canaus, sont relev6s par ordinateur, et les changements d'activit6 sont 6valu6s au moyen de statistique. On d6montre et ~tudie les changements d'activit6, dus aux rgactions des M6trifonate et Oxamniquine.
Ein mehrkanal-ultraschalI-Aktivit~itsbildschirm for i n - v i t r o abschirmung schistosomalhemmender Medikamente Zusammenfassung--Eine Mcthode zur Feststetlung der Wirkung schistosomalhemmender Medikamente auf die T~itigkeit der Schistosomen in vitro wurde erarbeitet und ausgewertet. Die Methode erl~iutert T/itigkeitsaufzeichnungen, der dutch die Wurmbewegungen erzeugten Unregelm~iBigkeiten in einem 10 MHz Ultraschallfeld. T~itigkeitsaufzeichnungen von bis zu 10 Kan~ilen werden von einem Computer aufgezeichnet und die T~itigkeitsver~inderungen werden zufriedenstellend ausgewertet. Es werden die T~itigkeitsver~inderungen in bezug auf Metrifonate und Oxamniquine dargestellt und diskutiert.
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July 1978
A multichannel ultrasonic activity monitor for in.vitro screening of antischistosomal drugs M . C. B r o w n Bioengineering and Medical Physics Unit, University of Liverpool, Liverpool L69 3BX, England
D. F. Norman
D. R, Bell
C . J . Chavasse
Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, England
A b s t r a c t - - A method of assessing the effect of antischistosomal drugs on the activity of schistosomes. in vitro has been devised and exploited. The system described records activity from the disturbances caused by the worm movements in a 10 MHz ultrasonic field, Activity recordings from up to 10 channels are plotted by computer, and changes in activity evaluated statistically. The activity changes in response to Metrifonate and to Oxamniquine are demonstrated and discussed
Keywords--Activity, Metrifonate, Oxamniquine, Schistosome, Ultrasonics
1 Introduction Bilharzia, is a parasitic disease of major medical and economic importance in the tropics. The symptoms are caused by large numbers of eggs laid by small worms (schistosomes) which live in the blood vessels of the gut or bladder. A major approach to the eradication of the disease is by chemotherapy to prevent the production of eggs, but the drugs which are currently available for this purpose are not ideal, either because of toxicity, lack of complete effectiveness, the need for multiple dosage, or cost. One possible way of evaluating antischistosomal drugs is to observe the effect they have on the activity of the worms maintained in vitro. The simple approach of visual observation has been used by COLES (1975) and other previous workers to obtain a qualitative idea of activity changes. Automated systems have been developed by HILLMAN and SE~qrx (1973) who detected the motion of the shadow cast by the worm, and by BROWN et al. (1973) using ultrasound. SCHISTOSOMIASIS, o r
2 Method The method employed here is a development of the ultrasonic method first described by BROWN et al. (1973). Each activity-monitoring unit uses a double ultrasonic transducer bonded onto the bottom of a glass vessel in which the schistosomes (approximately 10 mm long by 0" 5 m m diameter) are maintained in a flowing culture medium which can be modified by the addition or removal of the drug under test. The standing wave pattern of the ultrasound Received 12th August 1977
field inside the vessel is modified every time a worm, or part of a worm, moves, so causing a transient amplitude change in the sound level detected by the receiving transducer. Electrical signals corresponding to these amplitude disturbances are extracted by synchronous detection. F o r continuous worm movements, the signal from the detector is similar to that from Doppler velocity detection systems. The generation of activity signals may be clarified by reference to Fig. 1. The worm is considered here to be made up of small segments each reflecting the same ultrasonic energy and moving with a velocity component 11, away from the ultrasound transmitter/ receiver unit. The receiving ultrasonic transducer will detect a signal reflected from each segment which will have the form
where A is the reflected ultrasonic signal amplitude received from each segment, co is the ultrasonic angular frequency, c is the velocity of sound in the culture medium, 11, is the vertical velocity component of segment, t is the time, in seconds, and 0, is the phase angle of reflected signal from segment at t = 0. F o r the whole of the worm, segments 1 to N, the received signal will be 1N
F(t) = ~ A sin
[(
2V, c o ] ] co+ ~ / t + 0 , , /
V.+c]
j
This ultrasound signal, when amplified and detected synchronously by mixing with the transmitted
0140-0115/78/0755-0000 $1 950/0 9 IFMBE : 1978
408
Medical & Biological Engineering & Computing
July 1978
signal, has low-frequency electrical components of the form
E(t) = ~ K , A s i n [ (2V,,co) t+(O.-Or)
L V.+c
W h e r e K x is the amplification of preamplifier and Or is the reference phase of transmit signal. In our system this signal E(t) is amplified and bandpass filtered to select a band of frequencies corresponding to a limited range of velocities. Thus, the activity signal can be represented.
N
(2V, o~)
. . . .
E'(t) = 2a/rx2A Sln 1
_ . ~ V.+c
t
where 0.01 < V, < 1.0 mm/s and K2 is the combined amlolication factor including bandpass filter. The particular velocity range was selected after signal and noise measurements on narrow frequency bands during a period of typical worm activity. The lower cut-off frequency is also required as a d.c. block. A figure for activity is derived from E'(t) by taking the mean modulus over a period less than the sampling intervals of the logging system. This is achieved by a rectifier and an analogue averager. This arrangement is seen on the block diagram in Fig. 2.
3 Apparatus A culture system is required to supply the worms with nutrients and with drugs. A continuous-flow system rather than an intermittent irrigation system was chosen because of the desire to leave the worms undisturbed except for the experimental agents. However, intermittent flow systems have been used
////
/ ///
VELOCITY
INCIDENT AND If' REFLECTED ULTRASOUND
by others, such as SENFTet al. (1973). The fluid-flow system employed here is that shown in diagram form in Fig. 3. Unsteady flow caused by pump peristaltic action, motor noise or movements of the tubes are interpreted by the apparatus as worm activity since the worms are displaced by sudden changes in flow rate. Disturbances in the flow are partially damped by the use of soft narrow-bore feed tubes. Also, the roller pump was chosen for its relatively smooth action. The rate of fluid flow is governed by the rate required for nutrition, the cost of the culture fluid and the time taken for the test drugs to reach full concentration at the worms. The culture vessel itself is a U-shaped Pyrex glass tube of 6 mm o.d. and 3 m m i.d., and the worms normally remain at the bottom of the U-bend, in the ultrasonic field. Fig. 4 shows a detail of this. The transducers are PZT4 material cemented onto a flattened section on the Pyrex U-tube and the piezoelectric element is scribed on the back to produce three receiving and three transmitting elements, as shown in Fig. 5, with the intention 0f obtaining sensitivity to movement over an adequate length of the culture vessel. The actual field pattern inside the vessels is, of course, impossible to standardise exactly, but bonding the transducer to the vessel removes the variability of temporary coupling mechanisms used with unbonded transducers, and reduces microphony due to microscopic motion between the transducer and the vessel. The 10 MHz drive signal to the transmit elements in the transducers is derived from a t.t.l, crystal oscillator and amplified by 10 independent drive amplifiers, each with controllable output. The receive elements are coupled to a l0 MHz tuned amplifier and diode detector. At the detector the breakthrough signal from the transmitter is so large
VECTORS
(Vr~)
Fig. 1 Simplified visualisation of the interaction o f elemental worm seEments with the ultrasound beam. The worm is represented as lying in the path o f a parallel ultrasonic beam and is in an aqueous culture medium
I
Medical & Biological Engineering & Computing
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409
The actual time constant used is important since the voltages existing at the output of the averagers are time weighted by worm activity occurring in the immediate preceding period. Also s o m e element of the previous voltage sample will be present in each value. This error in the signal must be kept small or removed in the computer program. The data recording system used is an autoranging lO-channel digital voltmeter which records the time, channel numbers, and voltage samples from the output points on the averagers onto magnetic cassette tape. When the averager time constants are set to 30s, the voltages are normally sampled at 1 rain intervals. The data has been transferred to punched paper tape before filing on the Liverpool University ICL 1906S computer and programs have been developed to convert the readings into a logarithmic form and to produce logarithmic plots. Also by selecting time blocks, histograms of the amplitudes of readings can be produced to compare different periods (blocks) during the experiments. Using these same time
that synchronous detection occurs and the output contains the motion information. The relevant frequency components are selected for amplification by a d.c. block and h.f. cut with 3 dB points at 0-16 Hz and 14Hz. All these sections are housed close to the transducers inside a heated room. The signals are then transferred to the main equipment console in an adjacent room and applied to a true rectifier circuit to provide a unidirectional signal for determining the modulus. The signals from this point in the processing are displayed on a small oscilloscope adjacent to the experiment, and also on a large display in the recording room. Fig. 6 shows some typical displayed signals for two channels, The average amplitudes of these signals are obtained by an analogue averager (r = 30s) to remove short-term changes and produce a d.c. voltage which represents the average amplitude of the activity signal during the previous 30 s. The time constant of the averager circuits can be changed to suit the rate at which activity is to be sampled by the logging system, CULTURE VESSEL
lo..
Fig. 2 Block diagram of one channel of the monitoring system
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DATA RECORDING SYSTEM
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Medical & Biological Engineering & Computing
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blocks, mean and standard-deviation figures are produced for statistical consideration. 4 Results
Some of the earlier results have been used to fix the parameters of the present system. On recording activity by this method it is found that the sampled voltages may vary by two orders of magnitude (40 dB) over a period of 1-2 h. Variations between consecutive minute samples may be up to 20 dB although they are generally much less. When the worms are disturbed, activity may change by 40-80 dB in a very short time. Logarithmic presentation was therefore essential, but it has also proved to be appropriate to the behaviour patterns exhibited. In stable periods of
undisturbed activity the distribution of logarithmic values is near normal about the mean. Also, small induced changes in activity show good proportionality to the starting activity, suggesting that the basic activity pattern is logarithmic. The frequency spectrum of the Doppler signal (at the detector) was tested to find the correct bandpass characteristics for the amplifiers and Fig. 7 shows a result for a short period of recording. Maximum signal to noise ratio (and maximum signals) occur at very low Doppler frequencies, corresponding to worm movements which would be barely perceptible without a microscope. (A Doppler frequency of 1 Hz corresponds approximately to 0- 075 mm /s). We therefore decided that only frequencies below 14 Hz be included in the activity readings to restrict the noise bandwidth. It is important, however, when analysing the signal, to remember that no velocity information is included in the activity readings. 4.1 Sample experiments
Fig. 4 The culture vessel showing the bonded transducer and worm
Fig. 8a shows the computer activity plot recorded from two pairs of worms which were used as one of the controls for the experiment of Fig. 10. After approximately 2"5 h of recording, beginning immediately after removal from a mouse, the source of culture medium was changed (point X). After a further 2 h the worms were removed from the culture vessel (point Y) for microscopic examination, to establish a background noise level for the ultrasonic monitoring system. The activity plot is set out on a logarithmic scale based on l ' O m V output from the monitoring system and consists of samples recorded at 1 min intervals. Experimental and control graphs are
GLASS
Fig. 5 Section of transducer showing construction. The piezoelectric element is bonded on to a flat area ground on the base of the culture vessel. The piezoelectric element is 8 X 5 mm and the silvering on the back is scribed to make six narrow elements which are connected up as alternate transmit and receive transducers
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Medical & Biological Engineering & Computing
July 1978
411
normally presented superimposed, but are separated here for clarity. In this test the worms had a high activity when first removed from the mouse but became less active over the first hour. This is the usual behaviour.
Fig. 6 Activity signals seen on main display unit connected to the output of the true rectifier circuit. Sweep speed was 3 cm/s, and all signals are negative
After the first hour activity shows a relatively steady average activity with short-term variations of 6-10 dB. After the change to the second source of culture medium the mean activity looks slightly higher. This may be due to a very small difference between the two sources of culture media but is found to be statistically insignificant. After removal of the worms the background noise level is seen to be more than 30 dB below the previous mean activity level. To quantify these results, time blocks are selected during each phase of the experiment. Blocks are selected which have a steady mean activity, and
where possible after the transient effects on activity caused by interference with the apparatus or change in the culture medium have subsided. Here blocks AA and BB are the last hour before the next change in each case. The activity recordings within the selected time blocks are processed and presented as shown on Figs. 8b and 8c. A histogram of the frequency of activity samples falling within 2 dB bands gives a view of the level and spread of the samples. The calculated mean and standard deviation is also presented. The mean and standard deviation for the noise level is given in Fig. 8 caption. An indication of the real change in activity between blocks AA and BB can be gained from a simple comparison of the means. The difference in the means is 0"142 on the log scale. However, the application of a null hypothesis test (students t test) shows that p g 0" 05 suggesting that the activity may be unchanged. Fig. 9 shows an experiment of similar format but employing an organophosphorus compound (Metrifonate 10-4M) in the culture medium introduced at point X. Metrifonate is used to treat Schistosomiasis and is known to depress motor activity. The activity before the introduction of the drug did not stabilise and is therefore difficult to quantify. However, after point X there was a marked reduction in activity by approximately 60 dB. The output voltages after point X are near to the noise level of time block CC, and therefore represent minimal activity. The small difference between this suppressed
Fig. 7 Relative amplitude of the Doppler frequency components recorded from the detector using five worms, and then dead worms. The contribution from Doppler signals above 5 Hz is negligible. Also maximum signal to noise ratio of the overa# system (live worms against dead worms) occurs in the lowest frequency block
412
Medical & Biological Engineering & Computing
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activity and the noise level could be due to the activity associated with the living state of the
paralysed worms but it is m o r e likely that their presence in the culture vessel acts as an extra noise
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(a) Activity plot, using one minute sampling intervals and a logarithmic activity scale. Noise level is established with the worms removed at the end o f the test. The mean and standard deviations of the noise level are : M ---- O" 845, o" = 0 . 0 6 8 on the log scale using IV = 61 samples in block CC (b) and (c) The distribution of activity samples recorded in time blocks AA and BB, respectively, together with the mean and standard deviations J u l y 1978
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(a) Erratic but high activity of the fresh worms followed by an apparent total suppression of activity on introduction of the new culture medium containing the drug at point X. After a time the worms recovered 414
o
6
(b) and (c) The large difference between the activities before and after the introduction of the drug. The large spread in readings is caused by the change in the mean activity occurring during the two time blocks selected for comparison. The noise level established in block CC was : M = 1.022, (7 = O" 063 on the log scale for N ~ 41 samples
M e d i c a l & Biological Engineering & C o m p u t i n g
J u l y 1978
source in response to background vibrations of the apparatus, After about 30 minutes at this low level the activity recovered slowly through time block BB. This recovery may be caused by the worms overcoming the chemical blocking action of the drug or by hydrolysis
~
3,5
of the drug in solution (ARTHUR and CASIDA, 1957). The histograms of Figs. 9b and 9c show non-normal distributions of readings, which is attributable to the non-stationary nature of the mean activity in both time blocks. A different drug, oxamniquine, was used in the
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Medical & Biological Engineering & C o m p u t i n g
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Fig, 10Effect of Oxamniquine 7"16 x 1 0 - 4 M on one pa# of worms in a single channel (a) Erratic activity in the initial control phase is f o l l o w e d by a raised and very stable activity after introduction of the drug
I
c
(b) and (c) demonstrate these changes. The noise /eve/ established from block CC was: M = 0"627, ~T ----- O" 069 on the log scale for N = 61 samples
July 1978
415
experiment presented in Fig. 10. The two pairs of worms showed continuous changes in activity throughout the first phase, and after introduction of the new culture medium containing oxamniquine 7 . 1 6 x 10-*M, the activity is seen to rise sharply. The comparison of results is more clear on Figs. 10b and 10c. The wide spread of results in block A A is due to the movement of the mean. A most interesting effect of the drug, in addition to the increase in activity, is that the range of readings is confined to a much narrower band. We have noticed this short term effect of oxamniquine on other occasions. The change in mean activity between blocks A A and BB was 0-6333 on the log scale (p < 0.01). 5 Discussion The method described here for evaluating antischistosomal drugs may be questioned in three ways. First, is the ultrasonic method appropriate ? Secondly, how should 'activity' be defined? Thirdly, can 'activity' be usefully related to the effectiveness of the drugs ? Clearly the ultrasonic method has the drawback that the worms are exposed to the ultrasonic waves, creating an environment which is abnormal and possibly damaging. Total electrical power levels delivered to each transducer is of the order of 20 m W and the ultrasonic power in the sonicated zone will be rather less. It is not possible to quantify the actual sound intensity reaching the worms because of the complex shape of the vessels and the standing wave pattern. The worms, which are constantly moving in the ultrasound field, are assumed to absorb only a small portion of the total power. Some experiments were conducted involving sonication for a period of a week, after which time most worms die due to some deficiency in their environment, or delayed effects of the mechanical damage during initial removal from the mouse. N o difference in life span has been found between the sonicated and unsonicated worms maintained in otherwise identical culture conditions. Also, sonicated and unsonicated worms examined by electron microscopy show no obvious differences. Although it seems likely that the ultrasound must affect the results in some way, the effects do not appear to present serious problems. The range of activity levels discernible by the ultrasonic method is large and on occasions may exceed the dynamic range capabilities of the recording system. However, the sensitivity to small movements seems excessive at times because background vibration, even vehicles passing the building, sometimes register as activity. The most irritating feature of the ultrasonic system is its great sensitivity to the bubbles which form in the culture medium by outgassing, or from small leaks. These cause very high noise levels that register as activity which can be applied to the motion of these small worms. COLES 416
(1975) classified the worms as active, slightly active or dead. HENRY et al. (1976) also using visual observations defined four levels of activity. The two moving shadow detectors of HILLMAN and SENF~ (1973) and that of JEWSBURY et al. (1977) derive values for activity in different ways. In the apparatus used by HILLMAN and SENFT the shadow cast by the worm moves over a matrix of 19 optical fibre terminations. Photocells at the other ends of the fibres register the number of times the shadow passes. One activity count is registered for each time that any fibre is shaded. Their activity plot therefore represents, in a complex way, an index of how much the worm has moved in a horizontal plane, without differentiating movements back and forth from continuous translation. Because of the 19 discrete sensors and the digital mode of processing it can be seen that small movements may not be registered. The method has, however, been used extensively, and appears to give useful and meaningful results (SENFI and HILLMAN, 1973; HILLMAN et al., 1974; HILLMANet al., 1975). The moving shadow method of JEWSBURY,et al. (1977) is really a simplification of the method of HILLMAN and SENFT. A single light-dependent resistor (l.d.r.) replaces the multiple-fibre detection system. The l.d.r, element is already in the form of a grid, and is a suitable size to fit underneath the culture vessel. An indian-ink grid scribed on clear perspex is placed between the worm and 1.d.r. so that as the worm moves its shadow falls on different parts of the matrix of sensing elements formed by the scribed grid and the l.d.r, element. At present, the method of analysing the resistance variations caused by the movement of the shadow is to count the number of turning points on a chart record of resistance. The figure should correspond to the number of matrix squares covered and uncovered during the period under examination. In spite of a rather subjective analysis and likely linearity defects of the sensing system it appears to work well, and it is attractive because of its simplicity. Activity as recorded by the ultrasonic method of BROWN et aL (1973) is the time average of the amplitude of the ultrasound reflected from those parts of the worm which are moving within an accepted velocity band. The amplitude of the reflected sound depends largely on the area projected by the worms on the base of the culture vessel (the number of elemental segments as in Fig. 1). Unevenness in the field pattern, standing waves and uneven sensitivity of the receiving elements are of relatively small importance, as the worms will normally move throughout the sensitive zone. This argument can also be applied to the alternative methods. The signal available at the detector in each channel contains velocity information in the form of Doppler frequencies. However, it was not thought worthwhile to use this information since the power spectrum of the Doppler signal (Fig. 7) shows that there is little
Medical & Biological Engineering & Computing
July 1978
signal energy associated with the motion occurring at visible velocities. The more rapid (and visible) movements occur relatively infrequently and involve less of the length of the worm. Calibration of the response of the measuring system and defining an index of activity has been a difficult problem with all methods. During experiments, there is a datum level created by the mean activity of the worms before the addition of the drug, and a base line is provided by the noise level of the apparatus with the worms dead or removed. The use of a logarithmic scale for presentation and analysis has the added advantage here that the gain of the sensing and amplifying system is not relevant provided adequate signal to noise ratio is achieved. A problem experienced with the simple form of analogue averaging employed here is that consecutive readings taken by the data recording system are not entirely independent--a small proportion of the previous reading remains due to the time constant of the averaging circuit. This effect has been minimised by using a sampling period of not less than twice the average time constant and also by developing a computer program to remove the residual portion of previous readings. Considering the third question, of whether activity is really relevant in evaluating the performance of autischistosomal drugs, is more difficult. Antischistosomal drugs may be intended to kill the worms directly, paralyse them so that they are carried away by the blood, prevent them from mating, or prevent egg laying. Not all of these approaches would necessarily result in gross activity modification. We have already seen that activity may decrease or increase in response to drugs which are known to be clinically effective, and fortunately currently used drugs do appear to modify in vitro activity at concentrations corresponding to those achieved in the treatment of human patients. Our recordings, and those of other workers, usually show a change in activity when the new agents are introduced which can be seen clearly on the graphs. However, interpreting these changes quantitatively is more difficult. A major problem is that mean activity is not normally the same for more than a short period, and, in some cases, particularly with single worms, the mean is continually changing over a wide range, perhaps with some periodicity. So it is difficult to capture a period of stationary activity, and often impossible to attribute a change in activity to the new agent with certainty, without resorting to many repeats of the experiment. F o r experiments of more than one day, stationary statistics may be inadequate. There is obviously other information about activity which could be derived, such as the magnitude of frequency shift and the patterns of activity variation. However, these could be explored in the future. Medical & Biological Engineering & Computing G
6 Conclusions
The ultrasonic method of activity measurement, the signal processing and data processing procedures presented here have been applied successfully to small worms (schistosomes). The method has some attractive theoretical features and produces high resolution plots of activity during experiments on the effect of drugs on the schistosomes. Changes in the level and in the pattern of activity can be demonstrated. By comparison with alternative (optical) methods it appears that this method introduces more practical problems, but its resolution and the possibilities for statistical analysis are good. Variations in individual worm activity have been found to be large compared to some of the effects under study. Appendix Manufacturers Ismatec MP25F Autoanalyser pump Ismatec S.A. Limmatstrasse 109 8031 Zurich Switzerland
Ultrasonic Transducers Transducer Manufacturing Services Lauriston Cowbridge Hill Malmesbury Wiltshire SN16 9UI UK Data Logging System P.C.D. Ltd. Alpha Chambers Alexandra Road Farnborough Road Hampshire GU14 6BU UK
Acknowledgments This
work was conducted with support from the Edna McConnell Clark Foundation (Grant No. 276-4}013). The authors would also like to express their gratitude to Prof H. M. Gilles for providing facilities at the Liverpool School of Tropical Medicine. The samples of Metrifonate were kindly donated by Bayer A. G. Wuppertal-Eberfeld (Pt Nr. 9298/75) and the Oxamniquine by Pfizer, Sandwich, Kent, UK (lot no. 272-02). References ARTHUR, B. W., CASIDA, J. E. (1957) Metabolism and selectivity of O, O-dimethyl 2,2,2-trichlorol-l-hydroxyethyl phosphonate and its acetyl and derivatives, jr. Ag. & Food Chem. 5, 186-192. July 1978
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BROWN, M. C., KOURA, M., BELL, D. R., GILLES, H. M. (1973) An in vitro activity monitor for schistosomes: preliminary report. Ann. Trop. Med. & Parasit. 67, 3, 369-370. COLES,G. C. (1975) The response of Schistosoma mansoni maintained in vitro to schistosomicidal compounds. J. Helminth. 49, 205-209. HENRY, W., GIRGIS, N. I., MANSOUR, N. S. (1976) In vitro studies on the use of penicillamine with tartar emetic against Schistosoma mansoni worms. Ann. Trop. Med. & Parasit. 70, 425-429. HILLMAN, G. R., OLSEN, N. J., SENFT, A. W. (1974) Effect on Methysergide and Dihydroergotamine on Schistosoma mansoni. J. Pharmacol. & Exp. Therapeutics 188, 529-535.
HILLMAN, G. R., SENFT, A. W. (1973) Schistosome motility measurements: Response to drugs, ibid. 185, 177-184. HILLMAN, G. R., SENFT, A. W. (1975) Anticholinergic properties of the antischistosomal drug Hycanthone. Am. J. Trop. Med. & Hygiene 24, 877-884. JEWSBURY,J. M., HOMEWOOD,C. A., MARSHALL,I. (1977) Inexpensive apparatus for measuring activity of Schistosoma mansoni in vitro. Trans. R. Soc. Trop. Med. & Hygiene 71, 115.
SENFT,A. W., HILLMAN,G. R. (1973) Effect of Hycanthone Niridazole and Antimony Tartrate on schistosome motility. Am. J. Trop. Med. & Hygiene 22, 734-742.
Controle d'activitd ultrasonore, a canaux multiples, pour tri prealable, in vitro, des medicaments anti-schistosomes Sommaire---I1 d6crit une m6thode de mise au point et d'application in vitro, de l'effet de m6dicaments antischistosome sur l'activit6 des schistosomes. Le syst6me d6crit enregistre l'activit6 de troubles caus6s par les mouvements des vers, dans un champ ultrasonore de 10 MHz. Les enregistrements de l'activit6, allant jusqu'& 10 canaus, sont relev6s par ordinateur, et les changements d'activit6 sont 6valu6s au moyen de statistique. On d6montre et ~tudie les changements d'activit6, dus aux rgactions des M6trifonate et Oxamniquine.
Ein mehrkanal-ultraschalI-Aktivit~itsbildschirm for i n - v i t r o abschirmung schistosomalhemmender Medikamente Zusammenfassung--Eine Mcthode zur Feststetlung der Wirkung schistosomalhemmender Medikamente auf die T~itigkeit der Schistosomen in vitro wurde erarbeitet und ausgewertet. Die Methode erl~iutert T/itigkeitsaufzeichnungen, der dutch die Wurmbewegungen erzeugten Unregelm~iBigkeiten in einem 10 MHz Ultraschallfeld. T~itigkeitsaufzeichnungen von bis zu 10 Kan~ilen werden von einem Computer aufgezeichnet und die T~itigkeitsver~inderungen werden zufriedenstellend ausgewertet. Es werden die T~itigkeitsver~inderungen in bezug auf Metrifonate und Oxamniquine dargestellt und diskutiert.
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Medical & Biological Engineering & Computing
July 1978
