Acta Derm Venereol 1948;28(3):295- 324
By C. Lennhoff
The investigations, on which I am going to report, date in parts as far back as 20-25 years. I was obliged to abandon my notes pertaining to this work when fleeing from Norway, and they must be considered lost. Thus I must ask you to be indulgent if I cannot make definite statements on some points, and it is with particular regret that I find myself unable to give a detailed account of the experiments performed on animals without access to the experimental records. The fact that Prof. HELLERSTRÖM succeeded in arranging for microscopical specimens of stained spirochaetes previously prepared in Magdeburg to be forwarded from Oslo during the war was of supreme importance for the continuation of the work here in Stockholm.
In 1914 I had published, in Zeitschrift für Chemotherapie, a paper dealing with a method of demonstrating Tr. pallidum by means of arsphenamine. For this visualization of spirochaetes I made use the powerful reducing property of the arsphenamine. As reduction indicator I used a mixture of potassium ferricyanide and ferric chloride, from which Berlin blue develops on reduction, as well as silver nitrate solution. Even then I pointed out that especially clear-cut pictures of the spirochaetes are obtained when an ammoniacal solution - suspension of silver nitrate is substituted for the aqueous solution. After immersing a smear containing Tr. pallidum first in an arsphenamine solution and afterwards in a reduction indicator of this type the organisms are visualized. Subsequently these findings have been confirmed and amplified by other research-workers. - If arsphenamine is injected intravenously into a syphilitic rabbit and the serum taken from the chancre at short intervals is smeared on glass slides, which are treated with the above-mentioned mixture of potassium ferricyanide and ferric chloride, the spirochaetes will be found stained, and progressive morphological changes will be noted during their gradual disappearance. In my opinion, the immediate action of the arsphenamine upon spirochaetes was established for the first time by this experiment.
Furthermore, it was found that the spirochaetes of relapsing fever and anthrax bacilli can be stained with arsphenamine; the latter is a specific remedy for each disease. Simultaneously I reported that by using quinine microscopical demonstration is possible of malarial plasmodia by means of the thalleoquin reaction. In this connection it should be mentioned that in 1926, at the Mitteldeutscher Dermatologen-Kongress in Magdeburg, I was able to show in co-operation with Dr. KAGELMANN that the fungi of sporotrichosis can be made visible with iodine. In addition Tr. pallidum was made visible by using iodine and gold. Initially in co-operation with Dr. KAGELMANN, I devised a procedure for staining smears containing Tr. pallidum with mercury and bismuth. With arsphenamine and bismuth, but not with mercury, microscopical demonstration is successful if the films covering the slides are delicate. When working, with bismuth we used Nadisan and Neonadisan respectively, the bismuth absorbed by the spirochaetes being demonstrated by Legère's test or with ammonium sulphide. As regards mercury, a demonstration of the organisms in smears is successful only when working with thick-drop preparations, and in this case a colloidal layer of a certain thickness seems to be imperative. It is also necessary to use a dissociated mercury salt. At present I employ exclusively a 1 per cent aqueous solution of mercuric chloride. Further, it is important to maintain a temperature optimum of between 50-60°C. For this purpose, we used principally a paraffin thermostat in Magdeburg; here we use a water bath. At low temperatures the action of the mercury salt solution requires a considerably longer time; if the temperature optimum is exceeded, the stain becomes gradually weaker - identification being rendered impossible as far as I can recollect, at 92° C or 96° C. We made a special point of using a well saturated solution of ammonium sulphide.
Briefly, the procedure for staining spirochaetes in thick drops on glass slides is as follows:
Demonstration of spirochaetes in thick-drop and klatsch preparations.
I) Dry in air.
II) Immerse for 10 minutes at 56° C in 1 per cent mercuric chloride.
III) Pour off the fluid (do not rinse).
IV) Dry with filter paper.
V) Immerse for 20 minutes in well saturated solution of ammonium sulphide (preferably with the slide standing upright in a cuvette).
VI) Pour off the fluid.
VII) Rinse. Dry.
Optimal visualization will be obtained by following the above directions. If, however, it is only a matter of a practical demonstration of the spirochaetes, the periods of time specified, especially that of the ammonium sulphide treatment, may be considerably abbreviated. On the other hand, the specimens may quite well be exposed for several hours without injury at. 56° C to the action of the mercuric chloride solution.
From the time I spent in Magdeburg I still possess specimens, in which spirochaetes were demonstrated in thick drops of material taken from cases of tertiary syphilis. However, it was first here in Stockholm I recognized that this method of demonstrating Tr. pallidum is quantitatively superior to the dark-field technique, though of course it is no substitute for observing the organisms in motion. Here we met with 12 instances of primary lesions, in which no spirochaetes could be found on dark-field examination, whereas their presence was disclosed by the above procedure. In these cases we proceeded by first taking serum for dark-field examination, then for the thick-drop stain, and finally again for: darkfield examination. These latter examinations were carried out by at least 2, in a few instances even 3, different investigators, the result each time being negative. In spite of these negative findings, spirochaetes were observed in the thick-drop preparations, sometimes even in large numbers. In a case of secondary syphilis the result was identical. Out of 3 instances of lymph node aspiration on darkfield examination no spirochoetes were detected in 2 cases and a single spirochaete in 1 case, while on the other hand in the thick drop the organisms were easily demonstrated, sometimes in large numbers.l
Moreover, my researches here in Stockholm on this thick-drop procedure yielded results that were quite useful in yet another way. As you will hear presently, I was able to establish the presence of elements resembling spirochaetes in appearance in quite a considerable number of aetiologically obscure conditions. These spirochaetes are susceptible to staining if a glass slide is lightly touched with the dermal surface of an excised piece of skin, or with another tissue specimen, or vice versa (klatsch technique). In this manipulation pressure should be avoided, otherwise numerous fibrils and filaments are apt to be stained as well, which would render recognition of the spirochaetes difficult or, in occasional instances, actually impossible. The best results are obtained, e.g. in lichen ruber, if after contact with the specimen a droplet of tissue fluid remains on the slide. Naturally this klatsch procedure is not so conclusive for determining the pathogenicity of a given spirochaete as one that discloses the position of the organisms within the tissues. The technique, however, is simple and does not require much time. For demonstrating Tr. pallidum at an autopsy the method seems to be definitely useful.
Before proceeding to discuss the experiments performed on animals with bismuth and mercury, I should like to mention a preliminary investigation. If a rabbit is injected first subcutaneously with mercuric chloride solution, and immediately afterwards intravenously with a solution of sodium thiosulphate, a dark, smoky discoloration will presently appear at the site of the sublimate injection, due to the presence of an active sulphur compound, such as e.g. sulphuretted hydrogen, into which compound the sodium thiosulphate is rapidly turned even in the alkaline system. Many years after this experiment, during my stay in Oslo, I learned from a paper by OPPENHEIM that as early as in 1896 the pharmacologist FAUST of Vienna had by another method of approach arrived at the result that sodium thiosulphate is instantaneously transformed within the system. When he injected a lethal dose of potassium cyanide together with sodium thiosulphate into mice, the animals survived. If a syphilitic rabbit presenting a chancre teeming with spirochaetes is given an intravenous injection of sodium thiosulphate, the organisms will, on dark-field examination, have disappeared immediately, i.e. after the minimum time required for preparing the specimens. After some time spirochaetes may again be seen, thus showing - and this I ask you to bear in mind for the following discussion - that sulphur is an antisyphilitic, as had formerly been pointed out by other investigators as well; its curative action upon syphilis is, however, not a lasting one, i.e. if any conclusion at all may be drawn from the above experiment. If a syphilitic rabbit is injected intravenously with Nadisan or Neonadisan and subsequently with sodium thiosulphate, and then serum is taken from the chancre at short intervals, spirochaetes that are stained black will be found, the black stain being due to bisulfide having been absorbed by the organisms in the system of the rabbit. If my memory does not fail me, we have carried out corresponding experiments with Neonadisan and sodium iodate administered intravenously.
Experiments with mercury, which could only be carried out on rabbits, proved difficult. Obviously, there are certain differences between, on the one hand, the action of arsphenamine and bismuth and, on the other, that of mercury. I am, however, not able to give details.
If Salyrgan, a colloidal mercury preparation, is injected into the chancre of a rabbit and immediately followed by an injection of sodium thiosulphate into a vein, spirochaetes stained black will be found in the serum subsequently taken from the chancre. If my memory does not deceive me, it was recorded that we found spirochaetes showing the black stain as early as 2 minutes after the sodium thiosulphate injection. If a syphilitic rabbit is injected intravenously with Salyrgan and sodium iodide, then I seem to recall that the spirochaetes can be seen stained red, i.e. by mercuric iodide. Unfortunately, I am unable to give details of these experiments carried out so long ago, as pointed out in the introduction to the present paper.
The possibility of demonstrating spirochaetes with mercury has hitherto attained the greatest importance. With the assistance of Miss KOEHNE in Magdeburg, I had 'evolved a mercury impregnation method for spirochaetes situated in the tissues. Though as yet not free from considerable imperfections, it still seems capable of demonstrating not only the spirochaetes susceptible to the silver stains, such as Tr. pallidum, but in addition other spirochaetes hitherto unknown.
Before considering ,this method I will try to give you an idea of the reasoning that led me to it. The starting-point of my investigations constituted my endeavours to find an avenue of approach to the pathogenesis of psoriasis. Round about 1912, in Bern, I had demonstrated to JADASSOHN and some of my fellow-assistants some Giemsa-stained spirochaetes in a klatsch preparation made from a psoriasis cuticle. This occurred, as we later found out, about the same time as PROWAZEK, and also PELLER, of Nobl's clinic published their findings. Subsequently, during the first World War, I had carried out inoculation experiments on psoriasis patients, a report of which was published in Berliner Klinische Wochenschrift in 1920. Afterwards these inoculation experiments were continued and extended by Dr. KAGELMANN in Magdeburg; the results have not been published. In 1920, RASK reported to have found spirochaetes in psoriasis, which was universally rejected. However, although most of his observations fail to withstand criticism, a picture showing spirochaete-like elements within the epithelium is truly remarkable. In 1940, when staying in Oslo I ca1led in person on Dr. RASK and told him that, if he could describe -his method so that other workers would be able to repeat his demonstration of spirochaetes in the tissues, he would, in my opinion, have been the first to contribute material evidence of the pathogenetic significance of spirochaetes in psoriasis. Dr. RASK was unable, however, to make any statements as to his technique in addition to what he had published.
In 1921, at the Mitteldeutscher Dermatologen-Kongress in Halle, I had demonstrated a crystal violet stain which, owing to a misapprehension of my manuscript, was stated by HOFFMANN and HOFFMANN in Jadassohn's handbook to be unstable. Using this stain I also had found elements resembling spirochaetes in smears prepared from a psoriasis cuticle. However, since fibrin was also made visible with this method and since, in addition, it failed to yield information as to the position of the elements observed within the tissues, I regarded these findings as of minor importance. Yet when in Oslo, in 1940, I found again among my papers the sketches made from my preparations by Dr. KAGELMANN in 1920 or 1921, I felt that I had before me spirochaetes identical with those I had later demonstrated in the tissues. A series of examinations of psoriasis lesions for spirochaetes carried out with the conventional silver stains failed to give any particularly significant results.
Especially encouraging to me in my endeavours was the observation that a considerable proportion of psoriasis cases heal under treatment with mercurous iodide (the Hydrargyrosum jodidum of the Swedish Pharmacopoeia). This fact I had mentioned briefly in a discussion at the Leipziger Naturforschertag in 1922. To quote the expression I used on that occasion, »the trees will not grow skyhigh», that is to say, only in a proportion of the cases is a cure brought about, and relapses occur as with all the other methods; on the other hand, the number of cases cured by mercurous iodide exceeds that of the responses to arsenic. This therapeutical experience was supplemented by the discovery of the corymbose psoriasis, presented. by myself at a meeting of the Magdeburg Medical Society, and reported by KAGELMANN in Archiv für Dermatologie, vol. 146. These observations constituted a very strong incitement to attempt a demonstration of spirochaetes by means of mercury, in view of the parallelism between therapeutical action and microscopical visualization. Since I in co-operation with Dr. KAGELMANN succeeded in demonstrating Tr. pallidum with the thick-drop procedure, it seemed desirable to evolve a method of visualization in the tissues, which was done with the assistance of Miss KOEHNE.
As with silver staining, thin tissue specimens are immersed in concentrated solutions of mercuric chloride in absolute alcohol. Here in Stockholm we prolonged the alcohol treatment as compared with our previous procedure, the specimens now being kept for 6-9 months in this concentrated alcoholic mercuric chloride solution. Naturally, great care must be exercised in preventing the solution from evaporating during this long period. For this purpose the flasks are closed with well-fitting, paraffined corks and periodically examined for the formation of precipitates. Subsequent to the mercuric chloride treatment the specimens are rinsed in running water for 15-21 hours and afterwards immersed for 6-9 hours in an aqueous solution of ammonium sulphide. Here also the flasks must be tightly closed. Prior to being used.with tissue specimens, each fresh batch of the ammonium sulphide is tested as to its efficacy with thick-drop preparations of syphilitic chancre serum on glass slides. The reagent is then stored under paraffin in a refrigerator. It will now retain its applicability for at least a month. If stored for longer periods, the ammonium sulphide will deteriorate and assume a brownish tinge. The reagent must possess a satisfactory quality to ensure the success of the visualization. In Magdeburg the drug was ordered from a firm of chemical manufacturers, here it has been supplied to us of late by the Chemical Department of Karolinska Institutet. In order to ensure complete saturation of the ammonium sulphide solution, hydrogen sulphide is bubbled through an ammoniacal solution for 8-9 hours. After having been treated with ammonium sulphide the specimens are immediately immersed in absolute alcohol, which as a rule is twice changed, and as quickly as possible transferred into paraffin via xylene. If the specimens have been left in the mercuric chloride solution for too short a time, the mercury with which the spirochaetes are impregnated may be dissolved by the ammonium .sulphide, or the stain may be destroyed during the process of embedding.
In this connection I wish especially to point out a technical error I occasionally made here in Stockholm. As already emphasized, very thin slices of tissue should be taken for examination. At the out-patient department I had removed pieces of skin by shallow excision with a scalpel, subsequently spreading these upon a coverglass or a piece of filter-paper in order to prevent the margins from doubling up. Even if the specimens are separated from these supports after the lapse of only a few minutes, the staining of the spirochaetes in the dermis will be found to be less satisfactory, if not completely destroyed, within the areas that were in contact with the support. The specimens should therefore be transferred into the alcoholic mercuric chloride solution without employing a support of this type. By using a 5 % aqueous mercuric chloride solution instead a concentrated alcoholic mercuric chloride solution the period of treatment can be extended up to 3-6 months. With this modification, however, fibrils and filaments are apt to stain too, thus obstructing examination. Even the alcohol method fails to afford complete protection against the coincidental staining of fibrils. This applies particularly to specimens of facial skin undergoing senile degeneration,. and was especially noted when examining lesions of erythematodes. The 5 % aqueous solution has proved very useful in demonstrating spirochaetes in cases of eruptive fevers, particularly rubella, which had initially given rise to considerable difficulties. However, when a biopsy specimen, taken from a rubella patient on the first day of the eruption, was kept for 6 months in concentrated alcoholic mercuric chloride solution, i.e. longer than at the start of the investigations, the presence of spirochaetes was disclosed by this solution also. Attempts to accelerate considerably the action of the mercuric chloride solution by employing higher temperatures about 50° C., have resulted in a fairly satisfactory, and sometimes excellent, visualization of. the spirochaetes in a number of cases without producing consistently reliable results. I will, however, refrain from discussing these experiments. Even our alcoholic mercuric chloride procedure still needs improvement. A serious drawback with regard to skin lesions is that the spirochaetes situated within the epithelium are stained only in the marginal zones or in those areas where the epithelium is partly broken up by the morbid process. In some cases we have therefore scarified the epithelium with the scalpel; in Magdeburg - this .modification was not used by us in Stockholm - we had also achieved visualization in the epithelium by omitting to embed the specimens, Instead, these were dipped for a moment in distilled water subsequent to the mercuric chloride treatment. Frozen sections were then made which were stained on the slide with ammonium sulphide. In this procedure the sections should be carefully dried on the slides with filter-paper, as they otherwise are apt to fold up when the ammonium sulphide solution is applied. This procedure sometimes met with certain technical difficulties; in such cases it was modified as follows: The wet frozen section is placed upon a slide, which is laid, with the section facing downwards, on a cuvette-cover containing ammonium sulphide. Care is taken that the cuvette-cover is well closed by the slide. The period during which the solution, or vapours, of ammonium sulphide are allowed to act on the specimen, is 3-20 minutes. - Briefly summarized, our tissue procedure principally used at present is the following:
Demonstration of spirochaetes in tissue specimens.
I) Immerse the thinnest possible tissue pieces for about 6-9 months in:
Mercuric chloride 25.0,.
Absolute alcohol 75.0 (close flask with paraffined cork).
II) Rinse for about 15-21 hours in running water.
III) Immerse for about 9 hours in ammonium sulphide.
IV) After rapid transfer via alcohol and xylene, embed in paraffin. Frequently we employ counterstaining with haematoxylin and van Gieson's solution.
In addition to the examination of tissue specimens in Magdeburg, we attempted to demonstrate the spirochaetes of psoriasis, seborrhoic eczema, lichen ruber and pityriasis rosea with the aid of the dark-field procedure. As it is not possible in these diseases to obtain serum for dark-field examination in the same way as in syphilis, we evolved the following technique:
When dealing with psoriasis, seborrhoic eczema and pityriasis rosea, we collected a liberal amount of scales or, as was most often done in cases of lichen ruber, tissue material. The material obtained was ground in a sterile mortar, suspended in saline and - in order to remove coarser particles of tissue - strained through a single or double layer of sterile fine linen, occasionally also through a Seitz filter. The filtrate was centrifuged for 20-45 minutes at 3,000 revs/ min, and the supernatant fluid then drained off by means of a water-jet pump, with the exception of a minute amount covering the bottom of the tube and kept for dark-field examination. As regards psoriasis, I remember the figures quoted in the records: the number of spirochaetes found in a given case was small in spite of lengthy mostly 6-7, in exceptional instances only 2, and at the utmost 30. The organisms were coarse and moved in a characteristic, transversely wabbling fashion. According to the records, we have found spirochaetes in 92 of 96 psoriasis cases. with the procedure described..
I am unable to recall the actual figures for seborrhoic eczema, lichen ruber and pityriasis rosea. As far I know, we examined about 15 cases each of these diseases with the darkfield technique, with positive findings in 12-14 cases of each disorder. In these examinations we were unable to distinguish between the spirochaete of seborrhoic eczema and that of psoriasis. In accordance with its appearance in stained sections, the spirochaete of lichen ruber is delicate, and moves gracefully, i.e. without displaying the so-called »twisting» motion of Tr. pallidum. The spirochaete of pityriasis rosea moves with extraordinary velocity, often tumbling head over heels in its motion, and is in this state not recognizable as a spirochaete; when the organism has resumed its horizontal position however, its true character of a spirochaete will be revealed. Hitherto we have not been able to take up work with the tedious procedure of dark-field examination as no centrifuge has been available. However, we have carried out a few preliminary experiments using the simplified technique by scraping a small amount of tissue material from the dermal surface of the excised specimen into a drop of saline. Thus we were able to find spirochaetes, which in various instances were demonstrated to several gentlemen, in 13 cases of psoriasis, 5 of pityriasis rosea, 1 of seborrhoic eczema, 2 of lichen ruber, 3 of erythema exsudativum multiforme, and 1 of erythema nodosum.
Further, in Magdeburg, we had succeeded in growing the spirochaetes of psoriasis, lichen ruber and pityriasis rosea on culture media. The organisms were grown in a refrigerator at 5-8° C on two media introduced by REITER and HODER respectively; growth was arrested at 37° C; the optimal temperature was not ascertained. However, when dealing with other spirochaetes, the temperature most conducive to growth should generally be considered of interest, as this might prove capable of explaining the presence of particularly large numbers of spirochaetes in the skin, and of illuminating certain clinical aspects, e.g. in cases of eruptive fevers. When I was compelled to resign my appointment, the oldest psoriasis strain was at the 28th subculture. The transfers had been made at fortnightly intervals, the purity of the strain having been controlled in the 8th or 9th subculture. Cultivation had been carried out by Miss RILKE. Recently we resumed cultivation in a refrigerator in co-operation with Docent HOLLSTRÖM; hitherto we have obtained first cultures grown in HODER'S medium from cases of psoriasis and pityriasis rosea, also from those of erythematodes and erythema multiforme, the organisms of which have not been grown previously. In these experiments we used a medium prepared with rabbit-serum broth and rabbit liver thrice subjected to fractional sterilization (HODER, Z. Immun. Forsch. .1930).
Hitherto, I have mostly been concerned with demonstrating spirochates in tissue specimens, but I must refrain from discussing details. However, I wish to stress the fact that careful attention was paid as to whether or not the elements interpreted by us as spirochaetes were situated in loco morbi. Below, the term spirochaete will be used to design elements presenting the morphological aspect of spirochaetes, whether the living organisms have been studied in the dark-field or not. Firstly, I propose to give a tabulation of the diseases in question:
Psoriasis, seborrhoic eczema, lichen ruber, pityriasis rosea, erythematodes, erythema multiforme, erythema nodosum, zoster, varicella, morbilli, rubella, dermatitis herpetiformis, pemphigus, mycosis fungoides, parapsoriasis, lymphadenosis cutis benigna, lymphogranuloma inguinale, lymphogranulomatosis maligna, lymphogranulomatosis benigna, erythema migrans, acne necroticans, acne vulgaris, endocarditis and leucaemia.2
We have examined about 240 cases of psoriasis in all. In Magdeburg, on examination of skin sections, 72 out of 80 cases were positive, in Stockholm all the 52 examined cases were positive. This also applies to material obtained from Koebners phenomena. In addition, there were in Magdeburg 96 cases with dark-field examination, 92 of which were positive; in Stockholm we obtained 13 positive dark-field findings. When stained in the tissues, the spirochaete is as a rule rather coarse, with occasional delicate specimens. Therapeutically, psoriasis responds top spirochaeticides, arsenic and mercurous iodide, as I have mentioned earlier; furthermore gold, bismuth and iodine have been recommended for treatment. Following the administration of arsphenamine and gold, psoriasiform eruptions have been observed.
Of seborrhoic eczema altogether 27 cases were examined. Therapeutically, in addition to ammoniated mercury ointment sulphur is used principally; the latter is a spirochaeticide.
In lichen ruber another delicate spirochaete is present, which is distinguishable from that of psoriasis not only on dark-field examination but also in sections. In the spirochaete observed in cases of lichen ruber the coils are generally somewhat wider and more shallow than in Tr. pallidum. The characteristic spirochaete was demonstrated in about 60 cases in all, among which there was one showing occasional organisms in a Koebners phenomenon, and one organism was found in a lymph gland forwarded for examination by Docent HOLLSTRÖM. In addition, there were 2 further cases which, in my opinion, are of general significance. In each of 2 eruptions resembling lichen ruber, which had developed subsequent to the administration of arsphenamine and mercury respectively, I found spirochaetes identical with those otherwise observed in cases of lichen ruber. I addition to the lichen ruber caused by the use of arsphenamine and mercury, lichen ruber is also known to develop especially after chrysotherapy, as gold is also a spirochaeticide. Lichen ruber may be to be cured by arsenic, mercurous iodide and bismuth, i.e. by spirochaeticides, but may also be provoked by spirochaeticides. As is well-known, considerable credit is due to Milian for having pointed out, in general terms, the significance of the provocation of a latent microbism. Apparently this factor plays an especially important role, in the field of dermatology, as regards the spirochaeticides that simultaneously exert a curative action on the conditions concerned.
The total of pityriasis rosea cases examined amounts to about 80, practically all of them being positive. As a rule, the spirochaete is delicate in shape with narrow coils, both shape and size, however, being subject to considerable variations: there exist also fairly large and coarser forms, and organisms with quite shallow coils as well as such forming arcs and circles are comparatively frequent. Previously it had been contended by SAALFELD that pityriasis rosea is cured by arsenic; here it was established that a prompt cure results from bismuth and arsphenamine. A report on this observation will be given by Docent HOLLSTRÖM. the first to have treated pityriasis rosea with bismuth. Evidence is on record of pityriasis rosea being provoked by gold and arsphenamine.
Of erythematodes we have examined 60 cases in all, consistently with positive spirochaetal findings. In addition there are 3 cases, of which cubital lymph glands were forwarded for examination by Dr. THYRESSON. The lesions clear up after administration of gold, arsphenamine, bismuth and also mercurous iodide, as has been established by us here in Stockholm in experiments extending over a considerable period. According to MARTENSTEIN and GRANZOW the luetin reaction is positive in 75 per cent of the cases, i.e. as frequently as in tertiary syphilis. The Wassermann test is not infrequently positive in the acute cases, being positive in chronic cases if the amount of antigen is doubled. The relation between erythematodes and tuberculosis or streptococcal infection might possibly be interpreted on the basis of provocation of a latent microbism, which also applies to erythema nodosum. Again, the occurrence of tuberculosis subsequent to morbilli has been known for a long time. Parenthetically, I should like to mention that once in a case of erythematodes presenting symptoms of arthritis, I found spirochaetes in an endocarditic heart valve; I am unable to state whether in this case the endocarditis was due to the erythematodes, or was merely a case of (so-called rheumatic endocarditis). Furthermore, I once demonstrated spirochaetes in a valve in a case of rheumatic endocarditis, and twice in lymph glands. of patients suffering from rheumatic polyarthritis. I am not aware, nor have I investigated, whether salicylic acid arts as a spirochaeticide. I have examined 27 cases of erythema multiforme in addition to one case examined in Magdeburg. Among these, the findings were negative in sections in one case, doubtfully positive in another case, and positive in the remainder. One case of erythema multiforme with sparse nodular lesions following on the administration of sulfathiazole was positive. Besides salicylic acid, potassium iodide is recommended for treatment and, in relapsing cases, arsenic; possibly also provocation occurs brought about by salicylic acid, arsenicals and mercury.
8 cases of erythema nodosum have been examined (tissue specimens), with positive findings, and I demonstrated the organisms in the dark-field in one case to several colleagues. In addition, there were 7 cases of erythema nodosum after the administration of sulfathiazole, which were positive. As is well-known, MASSINI reported a long time ago that on darkfield examination in a case of erythema nodosum he had found spirochaetes which he was unable to stain whichever method he employed.
I have examined 34 cases of zoster, yielding positive results by me. Provocation has been described as being due to arsenic and its derivatives, potassium iodide, bismuth, mercury and Chrysolgan. Varicella infection from arsenic zoster has both been observed .and experimentally produced. Provided the virus is the same in both diseases, the transfer, of the arsenic zoster and its manifestation in the form of varicella may be considered to support the conception of the provocation of a latent microbism.
I have examined 15 varicella and 7 morbilli cases. In a case, in which Docent HOLLSTRÖM consulted me on the first morning of the morbilli eruption, I found four spirochaetes with peculiar, twisting movements in the blood after a dark-field search of about 45 minutes. SALIMBENI and KERMORGANI, of the Institut Pasteur, stated that they were able to culture at 32° C spirochaetes from morbilli blood collected prior to the development of the rash; they failed, however, to obtain a pure culture. As regards provocation, MILIAN has reported a case of morbilli after arsphenamine entailing infection of other persons.
I have examined altogether 13 cases of rubella. Prior to being able to examine sections, I had made a klatsch preparation showing definitely peculiar spirochaetes with which I was unfamiliar. 1 mentioned this at the time when demonstrating the preparation to Professor HELLERSTRÖM, Docent RINGERTZ and other gentlemen. This was the first and only instance that I seemed to have identified a new spirochaete solely on the basis of a klatsch preparation. In addition to the findings discussed above, I have met with a case of rubella after sulfathiazole and another one after arsphenamine, each of them positive. In the latter case typical lymphoglandular enlargement was noted in the occipital triangle. I am not aware of benign eruptive fevers having been treated with spirochaeticides. I was not afforded the opportunity examining cases of scarlet fever.
I have examined 7 positive cases of dermatitis herpetiformis. Also in this disease I consider provocation by spirochaeticides possible. There are cases in which the condition develops for the first time in previously healthy subjects after the administration of iodine or mercury and takes a course exactly identical with that observed in dermatitis herpetiformis, arising without any known excitive factor. As far as I know, there is no evidence of a reaction of supersensitiveness with such a course. Should it emerge on continued investigation that the local responses to iodine and mercury respectively are due to provocation, this observation would be of considerable interest.
I shall not discuss these conditions as I have examined only a few cases. The common feature is the therapeutical use of spirochaeticides. To my knowledge this is at present not known to apply to erythema migrans.3
I have examined 10 cases, of acne vulgaris but these were not exhaustively investigated; in the first place, the objection can be raised that positive spirochaetal findings might be due to a seborrhoic eczema conductive to the origination of the acne. Of lymphatic leucaemia I have collected 6 cases with positive findings, in one of which a positive klatsch preparation was obtained from bone marrow. Of myeloid leucaemia, there are 2 cases, one of them with a positive klatsch preparation of bone marrow. Therapeutically, arsenicals should be taken into consideration. Possibly also the leucaemoid reaction to mercury results from provocation.
Further more, I would like to mention that I have found spirochaetes in closed cancerous tumours; I do not propose, however, to discuss this observation until further thorough investigations have been made. 4
In one instance we found spirochaetes in a horny scale covering a soft naevus, and in another in a scale adhering to a lipoma, in neither case, however, in the tissues. Spirochaetes were not demonstrated in allergic eczema, Besnier's prurigo, lupus vulgaris, a case of glandular tuberculosis, mycotic lesions, a case of Recklinghausen's disease, nor in a case of pseudomyxoma peritonei and one of glomerulonephritis. These constitute some few controls, but naturally the range of control examinations must be considerably extended. At the present moment the observations indicate that spirochaetes are not present in all conditions, nor at all stages of their development.
In summarizing I should like especially to emphasize the following. On the basis of the parallelism between therapeutic action and microscopical visualization, a procedure was evolved whereby, with the aid of mercury. Tr. pallidum and new, unknown spirochaetes could be demonstrated. As to Tr. pallidum, the thick-drop procedure seems to be quantitatively superior to the dark-field technique, though naturally incapable of replacing the latter. The spirochaetes previously known are susceptible to the silver
stain, that is to say, they are argentum-positive; but it has been occasionally suggested that even this group includes argentum negative forms (HOLLANDE, HOFFMANN and KARRENBERG). The new, apparently very numerous spirochaetes are (generally?) argentum-negative. This is probably one of the reasons for their hitherto having escaped observation; another one is perhaps the fact that when dealing with the conditions in question, serum for dark-field examination cannot be obtained by so simple a procedure as that with which we are familiar in the examination of syphilitic lesions.
In the diseases, in which spirochaetes are present, treatment with spirochaeticides and provocation by the same seems to play an important part. Many observations suggest that toxicodermas presenting the aspect of idiopathic diseases are actually identical with the conditions they simulate.
It is generally known that identification of the spirochaetes depending entirely on morphological characteristics as displayed in the fixed condition is as a rule impossible; however, the spirochaete of psoriasis can be distinguished from that of lichen ruber even in tissue specimens. The new spirochaetes hitherto examined with the dark-field technique are distinctive for the various diseases. In Magdeburg the spirochaetes of psoriasis, lichen ruber and pityriasis rosea have been cultured in a refrigerator. Recently, in co-operation with Docent HOLLSTRÖM first cultures have also been obtained from psoriasis, pityriasis rosea, erythematodes and erythema multiforme. The organisms of both the latter diseases have not previously been grown.
As for the majority of the conditions examined, it is at present more or less likely that spirochaetes are of aetiological significance. In my opinion, as regards psoriasis, lichen ruber, pityriasis rosea and erythematodes, the aetiologic evidence for these being spirochaetoses is largely established; in the case of erythema multiforme I consider this highly probable. However, it should be strongly emphasized that as yet only the foundation is laid for further necessary investigations.
I have now come to the end of my thesis but before concluding I wish to express my deeply-felt gratitude to my former .associates. In the first place, my sincere thanks are due to Professor FELIX PINKUS, who after a long interval of involuntary leisure has given me highly valuable advice and assistance at a preliminary general survey of my previous specimens during our simultaneous stay in Oslo. From that period also dates my gratitude towards Professor VICTOR KAFKA, who has encouraged me by taking especially keen interest in my work. - Here in Sweden, I am first and foremost greatly indebted to Professor SVEN HELLERSTRÖM, who immediately welcomed me to his clinic and facilitated my work in every respect, as well as to the Prosectors of the Pathological Institute of St. Göran's Hospital, Dr. FREDRIK WAHLGREN and Dr. NILS RINGERTZ, who afforded me the opportunity of working in their institute and continually gave me their support. Furthermore, I proffer my thanks to Physician-in-Chief Docent Bo TARRAS-WAHLBERG and to all the other gentlemen who extended their kind help to me. However, in all probability I would not have been able at all to express my gratitude, had not Sweden received us refugees and afforded us the opportunity of working. To Sweden, therefore, our heart-felt thanks are addressed.
At the 11th Meeting of the Northern Dermatological Society, on June 9th, 1946 the following original preparations were demonstrated under the microscope:
I) Tr. pallidum, thick-drop preparation.
II) Spirochaetes in a papilla, psoriasis.
III) » in a vesicle, lichen ruber bullosus.
IV) » in the dermis, pityriasis rosea.
V) » in the epithelium, erythematodes.
VI) » at the lower epithelial margin, erythema multiforme.
VII) » in a lymph gland, myeloid leucaemia.
Author's address: Karolinska Institutets Dermatologiska Klinik, Karolinska Sjukhuset, Stockholm 60, Sweden.
When not stated that the pictures were taken from frozen sections, material embedded in paraffin had been used.
Table 1. - Figs. 1-3. - Drawings by Professor FELIX PINKUS. Compo oc. 4. Apochrom. imm. 2 mm. Figs. 4-23 are photomicrographs.
Fig.1. - Psoriasis: Spirochaetes in a horny scale..
Fig. 2. - Psoriasis: Spirochaete between parakeratosis and epithelium.
Fig. 3. - Psoriasis: Spirochaete beneath the stratum corneum.
Table 2. - Fig. 4. - Psoriasis: Spirochaete in the epithelium. Frozen section. X 600.
Fig. 5. - Psoriasis: Spirochaete in a papilla. X 600.
Table 3. - Fig. 6. - Psoriasis: Spirochaetes in the dermis. x 600.
Fig. 7. - Psoriasis: Spirochaete in a papilla (case from which the oldest culture was obtained). x 600.
Table 4. - Fig. 8. - Psoriasis: Spirochaete in a micro-abscess. X 1,000.
Fig. 9. - Pustular psoriasis: Spirochaete in a pustule. X 600.
Table 6. - Fig. 10. - Pustular psoriasis: Spirochaete, and perhaps spirochaetal debris, in a pustule. X .600.
Fig. 11. - Pustular psoriasis: Spirochaetes in a pustule. X 600.
Fig. 12. - Same area as in Fig. 11. X 1,000.
Table 6. - Fig. 13. - Psoriasis: Spirochaete In a vesicle wall. The black mass within the vesicle is precipitated mercuric sulphide.
Fig. 14. - Seborrhoic eczema: Spirochaetes adjacent to a hair follicle. X 600...
Table 7. - Fig. 15. - Lichen ruber: Spirochaetes in the epithelium. Frozen section. X 600.
Fig. 16. - Same area as in Fig. 15. X 1,000.
Table 8.- Fig. 17. - Lichen ruber: Spirochaete in an infiltrate. X 600.
Fig. 18. - Lichen ruber bullosus: Spirochaetes- in a vesicle. X 600.
Fig. 19. - Same area as in Fig. 18. X 1,000.
Table 9. - Fig. 20. - Pityriasis rosea: Spirochaetes in the epithelium. Frozen section. X 600.
Fig. 21. - Pityriasis rosea: Spirochaetes in the dermis. X 1,000.
Table 10. - Fig. 22. - Morbilli: Spirochaete in the dermis. X 600.
Fig. 23. - Zoster: Spirochaete in the dermis. X 1,000.
(pictures omitted – they were unfortunately photocopied too dark)
1 Addendum to proofs. Since reading this paper before the meeting, I have examined material obtained by lymph node aspiration in cases of various skin diseases. These finding. will later be reported elsewhere.
2 Addendum to proofs: In addition, granuloma anulare and bromoderma (?).
3 Addendum to proofs: subsequently 3 cases of erythema migrans received bismuth treatment, 2 of which with striking success; the third case, which is still under treatment fails to show a rapid improvement.
4 Addendum to proofs: Since the time of the Meeting the number of examinations has increased. I refrain, however, from giving data as to these continued investigations in correcting the proofs.