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Notes on the treatment of trypanosomiasis
14
THE TREATMENT
O F TRYPANOSOMIASIS.
BY PROFRSSOR B. M O O R E AN~ DBS. M. N I E R E N S T E i N A,~D T. L. T O D D , of the Liverpool School of Tropical Medicine,
(Read by Dr. M. NIERENSTEIN, Wedmesday,July lOth, 1907.)
THE therapeutics of the present time for trypanosomi~is can be divided into the following :-A. Arsenic Compo~nds.--l. Arsenious acid ; 2. Fowler's solution. B. Colo~ffs.--1. Colouring matters belonging to the diazo group, Ehrlich's trypanred and Mesnirs afrodol blue and afrodol violet; 2. Colouring matters belonging to the triphenyl-methan groups, Ehrlich's malachite green and fuchsin. C. Atoxyt, which being an organic compound of aniline and arsenious acid cannot be considered as merely "an arsenic compound," an expression recently used in the lay Press. The introduction of an acid radicle into an amido nucleus generally changes its basic character, and, of course, on the other hand, lessens the acidity. The combined effect of aniline and arsenic is, therefore, the resultant of these two factors: amido group and arsenious acid. We have been able to demonstrate experimentally that either the amount of aniline generally given in the form of atoxyl, or the arsenious acid contained in that drug, when administered alone, will kill an animal within a few hours after the injection. D. Combined Treatment.--1. Combination of arsenious or acid atoxyl with colouring matters, as trypanred, suggested by Thomas and Breinl, Laveran and Mesnil~ and
THE TREATMENT OF TRYPANOSOMIASIS.
1~
others; 2. Atoxyl and s t r y c h n i n e - - v a n Campenhout ~ 3. Atoxyl and sublimate. The last-named treatment has recently been worked out in the Liverpool School of Tropical Medicine, and the results obtained .on rats are very promising. We who have been working in the Liverpool School of Tropical Medicine found that in infected rats (T~njpanosama b ~ e / ) , one part of which were treated with atoxyl alone, and the remainder with atoxyl followed by mercury, the first lot generally had relapses in from sixeeen to twentyfive days, while a great number of the animals treated with atoxyl and mercury are still alive, some after a peirod of nine months. These results are certainly encouraging. In our treatment we used at the beginning to give high doses of atoxyl in order to drive ou~ the parasites entirely, and it was found that if sumcient atoxyl were administered the parasites (Trypanosoma brucci) would disappear in rats for from sixteen to twenty-five days. A disappearance of the parasites for a similar period was also observed in guinea-pigs and rabbits. Mercury was then given, and the treatment stopped. In the bigger animals this double treatment should be continued for some time. With regard to sleeping sickne~, we would like to suggest, as a routine treatment, that one cubic centimetre of a 20 per cent. solution of atoxyl should be given daily for one week, followed by one cubic centimetre of a 1 per cent. solution of sublimate four times. This treatment should be repeated and continued for an extended period, and, of course, may require oecasion~.l modification. It would perhaps be possible to give mercury in the form of pills. The theory of the action of mercury in this treatment has been discussed in a paper published in the Annals of
Tropical Medicine and Parasitology, of the I,iverpool School of Tropical Medicine, vol. i, No. 2. It has been found tha~ the toxic effect of atoxyl can be
16
THE TREATMENT OF TRYPANOSOMIASIS.
diminished by acetylating the atoxyh Such an acetylated atoxyl is an antipyrine which contains arsenious acid. Acetylated atoxyl has been shown to be of use in mice by Ehrlich, and in dogs by our experiments. We found, as a rule, that when atoxyl was given in sufficient quantity to drive out the parasites (nagana) in dogs the host was also killed, but using acetylated atoxyl we have been more or less successful. An interesting fact pointed out by Ehrlich is that the parasites can get used to a drug and become resistant. It is therefore advisable to alternate various trypanocidal agents in the treatment as much as possible, that is, to give atoxyl, some other trypanosomekilling drug, and mercury ; for instance, atoxyl, fuchsin, or some other colouring matter by the mouth, and sublimate. It has usuaJly been recommended to give atoxyl in high doses at the beginning. Whether or not this is of benefit is a question, as the atoxyl apparently accumulates in the body, and the toxic effect appears after a time. In laboratory experiments we have frequently noticed that even after the treatmex/t had been stopped for a week toxic effects appeared, and this could only be due to the fact that at~xyl had accumulated in the body, and the arsenic had been slowly broken up. We would like to refer to one experiment. A rat was treated about nine months ago with atoxyl and is still showing toxic effects. We think it necessary to point out that the administration of atoxyl should be given a very careful trial, but that extremely high doses should be avoided as much as possible. The action of atoxyl seems to be very much on the nervous system, as neuritis is frequently caused by the administration of this drug. Blindness has also been observed after the use of atoxyl, and the question therefore arises whether it would not be well to counteract somewhat the effect of atoxyl by making it less operative on the brain and nerves. This might be done by introducing radicles
THE TREATMENT OF TRYPANOSOMIASIS.
17
which are more fixed to the blood and less to the nervous system. W e think it necessary to draw attention to experiments which we have made in connection with the reappearance of trypanosomes after treatment with atoxyl. W e found that strains obtained from animals which had had relapses were sometimes much more virulent. Our ~gan~ strain, used for work generally, killed a rat in from five to seven days. This air,tinwas followed up, commencing on January lath until M a y 5th, by subinoculation from one rat to another. Four drops of blood were always used. In none of these rats was any sudden increase of virulence noticed ; but in experiments with the strain obtained from animals in which the trypanosomes had reappeared it was found that the experimental animal was killed in from three to five days. Out of seventeen rats used for these experiments, eleven showed a remarkable increase of virulence. Similar work done with Trypanosoma dimorphon showed the same increase of virulence. Our strain used to kill a rat in from fifteen to seventeen days, and we found that in four out of six strains obtained through relapses after treatment by atoxyl the virulence obtained was from eleven to nine days. W e have recently obtained through atoxyl a T~Tpanoso~a dimo~Thon strain which kills a rat in seven days. Similar work with Trypanosoma gambiense is now in progress. It was our intention not to publish the results of this work until we had satisfied ourselves that Trypanosoma gambiense behaves in a similar way, but since it is intended to commence atoxyl treatment on a large scMe in Uganda, we feel that a warning in this direction should be given. These observations emphasise the necessity for careful selection of " fly-free" sites for the separation camps and for some provision by which treated natives m a y be kept under observation for considerable periods. C
18
THE TREATMENT OF TRYPANOSOMIASIS.
The atoxyl and mercury treatment lately used in our laboratories gives, when compared with atoxyl, fairly permanent results, and if an atoxyl treatment should be undertaken in Uganda, it would be advisable to start at once with this combined treatment, even if the reliability of this double treatment has so far not been fully established. Summarising, we would like to suggest, in the treatment of sleeping sickness, a fresh 20 per cent. solution of atoxyl, warmed up to 40 deg., to be administered in small doses to commence with, and the doses to be gradually increased, not, if possible, passing the limit of one cubic eentimetre of a 20 per cent. solution. The atoxyl to be followed as soon as possible by mercury in the form of sublimate, and, in addition, some other trypanoeide to be given. We would especially recommend fuchsin. We have lately tried a large number of colouring matters, both Ehrlich's and Hesnil and Nicolle's, and have come to the conclusion that fuehsin is the most promising. In addition to this, as was insisted upon by Thomas and Breinl, the general strength of the patient must be sustained ,in every way possible.
Dr. ANTON BREINL, Director of the Runcorn Research Laboratories, described and demonstrated the morphology of T~ypanosoma gambiense, and the changes which it exhibits : 1. During reproduction; 2. During treatment of its host by atoxyl. The effect of the latter drug, he said, was to cause the parasite to secrete a capsule, and to then withdraw into the internal circulation. In the blood-vessels, reproduction by sexual conjugation did not take place; at least it was never found, but division, after distinctive changes in nucleus and blepharoplast, was very common. He further demonstrated a new method of making blood films by first spreading a layer of liquid albumin on the
19
THE TREATMENT OF TRYPANOSObIIASIS.
slide, which prevented shrinkage of the protozoal tissues when spirit was applied to stain or fix the specimens. By Flemming's solution, and the subsequent use of safranin, polychrome methylene blue, and orange tannin, details impossible to be seen by the older methods could be well made out. DR. TODD (Liverpool School of Tropical Medicine) read a paper " O n Some Protozoal Parasites found in Animals on the Congo." I-Ie described the phases seen in a leucocytozoon from t h e blood of a hawk, which had been described by Schaudinn, and of which male and female forms were seen. The host (a grey hawk, Asturin~la monogrammica, common on the Congo), harboured large numbers of parasites, but it was probable the name Leucocytozoon would have to be changed, as L. Ziemanni occurred in other than white cells, and it was not related to the Leucocytozoon of mammals, which was a true white.cell parasite. Prior to conjugation, the matured parasite became rounded, and the sheath of periplast was shed, with the effete nucleus of the leucocyte post-cell. There were resting and motile stages, as noted by Schaudinn. Nuclear material was abundant in the male, less so in the female. This chromatophilic structure passed through several definite stages in development, each of which had distinctive appearances, and these Dr. Todd described. The whole process was akin to the trypanosome stage of Halteridium noctux (Schaudinn). Dr. Todd said that among other protozoal parasites of amphibia, T~.ypanosoma loricatum was found in several species of frogs and toads. It was seen only in the blood, in which Drepanidium was almost~ always present, but there was no definite relation as to numbers. The division of T. loricatum was peculiar, and various forms, from gerpetomonas-like bodies to the origina ! typical trypanoseines, were to be seen; the whole process was continuous, 02
20
THE TREATMENT
OF TRYPANOSOMIASIS.
but full of interesting stages, e.g.,rotation in opposite directions of dividing cells,division int~ sixteen cells within the envelope of the trypanosomc, and subsequent development of a flagellum. Dr. Todd said that present methods of staining were insufficientfor protozoal work ; they showed the structures but no detail. The study of protozoa was widely separated from that of bacteria, and must be approached from a different basis, and with the knowledge which we already had that the life-cyclesof many species were highly complicated. Continuous observation of living parasites was essential, and parallel hypotheses, acquired by knowledge of malaria, were only useful as aids to interpretation. SIR PATRICK MANSON said the Society was greatly indebted to the members of the Liverpool School, who had, at considerable inconvenience to themselves, travelled to London to give them the excellent demonstrations and observations which they had listened to that evening. The development of piroplasma was of great interest and importance, and the researches of Captain Christophers would, he believed, throw much light on a problem in protozoology of which little had hitherto been known. At the request of Dr. Todd, Sir Patrick Manson described the resultshe had experienced in the treatment of Europeans affected with trypanosomiasis by atoxyl. Several cases under his care undergoing this treatment were doing well, and he founded great hopes on the method. Just as mercury was given in syphilis, so should atoxyl be administered in trypanosomiasis, namely, in small doses for prolonged periods. One case so treated was now free from fever, the patient's blood did not infect when inoculated into animals, and he was apparently cured. He regarded the clinical question as being in many ways parallel to that of syphilis, and said that with care, abundant nourishment, and treatment in the earlier stages,there was a moderately
THE TREATMENT OF TRYPANOSOMIASIS.
21
hopeful prospect of ultimately controlling the disease, or, at least, materially reducing the mortality. He further described at length the cases of other Europeans, and said that he agreed that atoxyl followed by mercury was a rational, and possibly would be found to be a successful, treatment.
THE TREATMENT
O F TRYPANOSOMIASIS.
BY PROFRSSOR B. M O O R E AN~ DBS. M. N I E R E N S T E i N A,~D T. L. T O D D , of the Liverpool School of Tropical Medicine,
(Read by Dr. M. NIERENSTEIN, Wedmesday,July lOth, 1907.)
THE therapeutics of the present time for trypanosomi~is can be divided into the following :-A. Arsenic Compo~nds.--l. Arsenious acid ; 2. Fowler's solution. B. Colo~ffs.--1. Colouring matters belonging to the diazo group, Ehrlich's trypanred and Mesnirs afrodol blue and afrodol violet; 2. Colouring matters belonging to the triphenyl-methan groups, Ehrlich's malachite green and fuchsin. C. Atoxyt, which being an organic compound of aniline and arsenious acid cannot be considered as merely "an arsenic compound," an expression recently used in the lay Press. The introduction of an acid radicle into an amido nucleus generally changes its basic character, and, of course, on the other hand, lessens the acidity. The combined effect of aniline and arsenic is, therefore, the resultant of these two factors: amido group and arsenious acid. We have been able to demonstrate experimentally that either the amount of aniline generally given in the form of atoxyl, or the arsenious acid contained in that drug, when administered alone, will kill an animal within a few hours after the injection. D. Combined Treatment.--1. Combination of arsenious or acid atoxyl with colouring matters, as trypanred, suggested by Thomas and Breinl, Laveran and Mesnil~ and
THE TREATMENT OF TRYPANOSOMIASIS.
1~
others; 2. Atoxyl and s t r y c h n i n e - - v a n Campenhout ~ 3. Atoxyl and sublimate. The last-named treatment has recently been worked out in the Liverpool School of Tropical Medicine, and the results obtained .on rats are very promising. We who have been working in the Liverpool School of Tropical Medicine found that in infected rats (T~njpanosama b ~ e / ) , one part of which were treated with atoxyl alone, and the remainder with atoxyl followed by mercury, the first lot generally had relapses in from sixeeen to twentyfive days, while a great number of the animals treated with atoxyl and mercury are still alive, some after a peirod of nine months. These results are certainly encouraging. In our treatment we used at the beginning to give high doses of atoxyl in order to drive ou~ the parasites entirely, and it was found that if sumcient atoxyl were administered the parasites (Trypanosoma brucci) would disappear in rats for from sixteen to twenty-five days. A disappearance of the parasites for a similar period was also observed in guinea-pigs and rabbits. Mercury was then given, and the treatment stopped. In the bigger animals this double treatment should be continued for some time. With regard to sleeping sickne~, we would like to suggest, as a routine treatment, that one cubic centimetre of a 20 per cent. solution of atoxyl should be given daily for one week, followed by one cubic centimetre of a 1 per cent. solution of sublimate four times. This treatment should be repeated and continued for an extended period, and, of course, may require oecasion~.l modification. It would perhaps be possible to give mercury in the form of pills. The theory of the action of mercury in this treatment has been discussed in a paper published in the Annals of
Tropical Medicine and Parasitology, of the I,iverpool School of Tropical Medicine, vol. i, No. 2. It has been found tha~ the toxic effect of atoxyl can be
16
THE TREATMENT OF TRYPANOSOMIASIS.
diminished by acetylating the atoxyh Such an acetylated atoxyl is an antipyrine which contains arsenious acid. Acetylated atoxyl has been shown to be of use in mice by Ehrlich, and in dogs by our experiments. We found, as a rule, that when atoxyl was given in sufficient quantity to drive out the parasites (nagana) in dogs the host was also killed, but using acetylated atoxyl we have been more or less successful. An interesting fact pointed out by Ehrlich is that the parasites can get used to a drug and become resistant. It is therefore advisable to alternate various trypanocidal agents in the treatment as much as possible, that is, to give atoxyl, some other trypanosomekilling drug, and mercury ; for instance, atoxyl, fuchsin, or some other colouring matter by the mouth, and sublimate. It has usuaJly been recommended to give atoxyl in high doses at the beginning. Whether or not this is of benefit is a question, as the atoxyl apparently accumulates in the body, and the toxic effect appears after a time. In laboratory experiments we have frequently noticed that even after the treatmex/t had been stopped for a week toxic effects appeared, and this could only be due to the fact that at~xyl had accumulated in the body, and the arsenic had been slowly broken up. We would like to refer to one experiment. A rat was treated about nine months ago with atoxyl and is still showing toxic effects. We think it necessary to point out that the administration of atoxyl should be given a very careful trial, but that extremely high doses should be avoided as much as possible. The action of atoxyl seems to be very much on the nervous system, as neuritis is frequently caused by the administration of this drug. Blindness has also been observed after the use of atoxyl, and the question therefore arises whether it would not be well to counteract somewhat the effect of atoxyl by making it less operative on the brain and nerves. This might be done by introducing radicles
THE TREATMENT OF TRYPANOSOMIASIS.
17
which are more fixed to the blood and less to the nervous system. W e think it necessary to draw attention to experiments which we have made in connection with the reappearance of trypanosomes after treatment with atoxyl. W e found that strains obtained from animals which had had relapses were sometimes much more virulent. Our ~gan~ strain, used for work generally, killed a rat in from five to seven days. This air,tinwas followed up, commencing on January lath until M a y 5th, by subinoculation from one rat to another. Four drops of blood were always used. In none of these rats was any sudden increase of virulence noticed ; but in experiments with the strain obtained from animals in which the trypanosomes had reappeared it was found that the experimental animal was killed in from three to five days. Out of seventeen rats used for these experiments, eleven showed a remarkable increase of virulence. Similar work done with Trypanosoma dimorphon showed the same increase of virulence. Our strain used to kill a rat in from fifteen to seventeen days, and we found that in four out of six strains obtained through relapses after treatment by atoxyl the virulence obtained was from eleven to nine days. W e have recently obtained through atoxyl a T~Tpanoso~a dimo~Thon strain which kills a rat in seven days. Similar work with Trypanosoma gambiense is now in progress. It was our intention not to publish the results of this work until we had satisfied ourselves that Trypanosoma gambiense behaves in a similar way, but since it is intended to commence atoxyl treatment on a large scMe in Uganda, we feel that a warning in this direction should be given. These observations emphasise the necessity for careful selection of " fly-free" sites for the separation camps and for some provision by which treated natives m a y be kept under observation for considerable periods. C
18
THE TREATMENT OF TRYPANOSOMIASIS.
The atoxyl and mercury treatment lately used in our laboratories gives, when compared with atoxyl, fairly permanent results, and if an atoxyl treatment should be undertaken in Uganda, it would be advisable to start at once with this combined treatment, even if the reliability of this double treatment has so far not been fully established. Summarising, we would like to suggest, in the treatment of sleeping sickness, a fresh 20 per cent. solution of atoxyl, warmed up to 40 deg., to be administered in small doses to commence with, and the doses to be gradually increased, not, if possible, passing the limit of one cubic eentimetre of a 20 per cent. solution. The atoxyl to be followed as soon as possible by mercury in the form of sublimate, and, in addition, some other trypanoeide to be given. We would especially recommend fuchsin. We have lately tried a large number of colouring matters, both Ehrlich's and Hesnil and Nicolle's, and have come to the conclusion that fuehsin is the most promising. In addition to this, as was insisted upon by Thomas and Breinl, the general strength of the patient must be sustained ,in every way possible.
Dr. ANTON BREINL, Director of the Runcorn Research Laboratories, described and demonstrated the morphology of T~ypanosoma gambiense, and the changes which it exhibits : 1. During reproduction; 2. During treatment of its host by atoxyl. The effect of the latter drug, he said, was to cause the parasite to secrete a capsule, and to then withdraw into the internal circulation. In the blood-vessels, reproduction by sexual conjugation did not take place; at least it was never found, but division, after distinctive changes in nucleus and blepharoplast, was very common. He further demonstrated a new method of making blood films by first spreading a layer of liquid albumin on the
19
THE TREATMENT OF TRYPANOSObIIASIS.
slide, which prevented shrinkage of the protozoal tissues when spirit was applied to stain or fix the specimens. By Flemming's solution, and the subsequent use of safranin, polychrome methylene blue, and orange tannin, details impossible to be seen by the older methods could be well made out. DR. TODD (Liverpool School of Tropical Medicine) read a paper " O n Some Protozoal Parasites found in Animals on the Congo." I-Ie described the phases seen in a leucocytozoon from t h e blood of a hawk, which had been described by Schaudinn, and of which male and female forms were seen. The host (a grey hawk, Asturin~la monogrammica, common on the Congo), harboured large numbers of parasites, but it was probable the name Leucocytozoon would have to be changed, as L. Ziemanni occurred in other than white cells, and it was not related to the Leucocytozoon of mammals, which was a true white.cell parasite. Prior to conjugation, the matured parasite became rounded, and the sheath of periplast was shed, with the effete nucleus of the leucocyte post-cell. There were resting and motile stages, as noted by Schaudinn. Nuclear material was abundant in the male, less so in the female. This chromatophilic structure passed through several definite stages in development, each of which had distinctive appearances, and these Dr. Todd described. The whole process was akin to the trypanosome stage of Halteridium noctux (Schaudinn). Dr. Todd said that among other protozoal parasites of amphibia, T~.ypanosoma loricatum was found in several species of frogs and toads. It was seen only in the blood, in which Drepanidium was almost~ always present, but there was no definite relation as to numbers. The division of T. loricatum was peculiar, and various forms, from gerpetomonas-like bodies to the origina ! typical trypanoseines, were to be seen; the whole process was continuous, 02
20
THE TREATMENT
OF TRYPANOSOMIASIS.
but full of interesting stages, e.g.,rotation in opposite directions of dividing cells,division int~ sixteen cells within the envelope of the trypanosomc, and subsequent development of a flagellum. Dr. Todd said that present methods of staining were insufficientfor protozoal work ; they showed the structures but no detail. The study of protozoa was widely separated from that of bacteria, and must be approached from a different basis, and with the knowledge which we already had that the life-cyclesof many species were highly complicated. Continuous observation of living parasites was essential, and parallel hypotheses, acquired by knowledge of malaria, were only useful as aids to interpretation. SIR PATRICK MANSON said the Society was greatly indebted to the members of the Liverpool School, who had, at considerable inconvenience to themselves, travelled to London to give them the excellent demonstrations and observations which they had listened to that evening. The development of piroplasma was of great interest and importance, and the researches of Captain Christophers would, he believed, throw much light on a problem in protozoology of which little had hitherto been known. At the request of Dr. Todd, Sir Patrick Manson described the resultshe had experienced in the treatment of Europeans affected with trypanosomiasis by atoxyl. Several cases under his care undergoing this treatment were doing well, and he founded great hopes on the method. Just as mercury was given in syphilis, so should atoxyl be administered in trypanosomiasis, namely, in small doses for prolonged periods. One case so treated was now free from fever, the patient's blood did not infect when inoculated into animals, and he was apparently cured. He regarded the clinical question as being in many ways parallel to that of syphilis, and said that with care, abundant nourishment, and treatment in the earlier stages,there was a moderately
THE TREATMENT OF TRYPANOSOMIASIS.
21
hopeful prospect of ultimately controlling the disease, or, at least, materially reducing the mortality. He further described at length the cases of other Europeans, and said that he agreed that atoxyl followed by mercury was a rational, and possibly would be found to be a successful, treatment.