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A LABORATORY INFIECTION WITH THESPuROTRICHUMI DE BEURMANNI

Centralblatt fUr Bakteriologie H. Fielitzund Parasitologie(Central Journal of Bacteriologyand Parasitology)Vol. 55 pages 361-370 [see Note 1)19 10

Since the discovery of sporotrichosis by B.R. Schenckand its thorough study by de Beurmann this disease has beenrepeatedly reported on in America, but especially frequentlyLnd down to very recently in France, while the few observa-tions in other countries have remained scattered up to thepresent. The first case of sporotrichosis in Germany, whichwas observed toward the end of last year at the Royal Univer-sity Polyclinic for Skin Diseases and Venereal Diseases atBerlin L2] has not been followed by a second, although severalarticles concerning sporotrichosis have appeared in the Germanliterature and the attention of large numbers among us hasthus been directed toward this interesting and practically im-portant disease.

[Note 1] 1 have already reported briefly on this infec-tion in the Berlin Medical Society on 4 May of this year.

[Note 2) Arndt, G., "A Contribution to Knowledge ofSporotrichosis of the Skin, with Special Attention to Lymph-angitis Sporotrichosica. Experimental Sporotrichosis," Der-matologische Zeitschrift (Dermatological Journal), Vol 17,No I and 3.

It thus appears that the infrequency of observations onthis subject is due not only to the extraordinary difficultyof diagnosis (confusion with tuberculosis and lues!) but ratherto the uneven distribution of this disease. Indeed, in our ownpolyclinic, in spite of the vast field of observation and thefact that our attention was particularly directed toward it,no new observation has yet been made.

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The case on which I am r•portinri In this article doeszvt represent & genulne sporotrichosisr b*At a laboratory in-fection wqI incurred m44"", probably i-n inoculation ofanimals or^I dissecting animals diseased with sporotrichosisor perhaps in sporoagglutination experiments.

The strain of the fungus which was used in these ex-periments was derived from the above-mentioned case, and myinfection like the other ran the course of the regionallyrestricted sporotrichosis, but did not reach the same stageof advanced development (lymphangitis covering almost the en-tire length of the arm, with multiple abs-'esses), because itsnature was correctly identified soon after it set in and itwas appropriately treated.

The only skin injury that I am aware of and that couldboth from the time of its occurrence and from its locationcome into question as the place of entry of the virus was onethat I inflicted on myself on 23 December 1909 for the purposeof an inoculation in experimental work with mollusc material.Before the inoculation the skin on the flexor side of theright forearm, approximately in the middle, in an area wherethe sporotrichotic inoculation focus later developed, wasrubbed with Dumice, so that at the place most affected a vesi-cular dermatitis developed, which disappeared without a tracewithin two weeks under treatment with powder and sealingbandages.

If this skin injury was causally connected with the in-fection, the first clinical symptom, which appeared early inMarch of this year, must have been preceded by an incubationperiod of about two months.

The disorder first manifested itself in a barely lentil-sized red, rather hard, non-painful, hemispherically raisedlump on the flexor side of the right forearm, which within twoweeks increased to the size of a bean, became soft in the cen-ter, and was covered with a brownish crust, after the removalof which an abscess became visible. With the softening of theprimary focus a somewhat painful swelling of the right elbowand axillary glandeset in. Hard, pressure-sensitive strandsalso formed under the skin between the abscess and the swollenelbow gland.

On 23 March 1910 the following condition was found:

On the flexor and radius side of the right forearmmore or less in its center there was a not quite round, flatabscess about the size of a one-pfennig piece, surrounded bya bluish-red, somewhat wall-like border about 0.5 cm wide,here and there somewhat undermined; the base of the abscessconsisted of a smooth, yellowish, area of decayed flesh, verypainful to the touch, covered with a clear, thin, yellowishbrown fluid and showing a few granulations as big as the headof a pin or a little bigger (Photograph no. I).

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GRAPHIC NOT REPRODUCIBLE

Photograph I. The inoculation sore on the fifthday after its inception, before beginning of thepotassium iodide treatment; slight redness andscaling of the skin in the vicinity of the soreare remains of an iodoform dermatitis.

The lower stratum of the abscess is taken up by a hardinfiltrate extending into the deeper layers of the skin,shading off into the healthy surrounding area without sharpoutlines and freely movable with the skin over the tissuesbeneath.

Above the right condylus internus humeri a rather hardgland about the size of a bean can be felt under the skin.In the right underarm is a gland of the same consistency andabout the size of a hazelnut. Both glands are slightly sen-sitive to pressure.

Swollen lymph glands are not visible externally. Sub-jectively, however, when the arm is extended a slight pain isfelt running from the abscess to the swollen gland in theelbow. Upon close examination two hard strands can be founddeep in the skin, connecting the abscess with the elbow gland,one in a straight line, the other in a slight curve with theconvexity toward the ulnar side of the forearm. Both strandsexhibit numerous nodular inspissations from the size of alentil to that of a pea, in some cases in a row like a stringof beads; some of these raise the skin visibly.

Over the swollen glands and over the inflamed lymphvessels the skin is freely movable and not discolored.

The general state of health is not affected in any way.

The clinical picture showed nothing specifically charac-teristic, and admitted of the following interpretations:

Apart from an abscess formation in connection with anordinary pus coccus infection, a prominent possibility wasulcus molle; the acute course, the shape and boundary of theulcer, the character of the floor of the ulcer, and most

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especially the simultaneous occurrence of a painful swelling-of the corretoonding lymph glands were clinically completelyconsistent wit'l ulcus molle. Although examination of the pusand granulationý. for streptobacilli gave a compleLely negativeresult, the ulcer was cauterized with carbolic acid andsprinkled with iodoform. This therapy had no considerableeffect, and had to be broken off a,'-ain immediately because ofa vesicular dermatitis that ensued.

There was also sowe question of a tuberculous ulcer,possibly originating through inoculation. Repeated examina-tions for tuberculosis bacilli, which are usually easy to findin acute cases of inoculation tuberculosis of the skin, turnedout negative.

For safety's sake the serous secretion was also testedfor syphilis spirochetes, althouGh the ulcer showed no simila-rity to a primary effect and no secondary or tertiary lueticcould have been present because of lack of a prior luetic in-fection.

While chronic glanders of the skin can produce symptomssuch as were present here, it could be ruled out in advance,because I had not come into contact with material containingglanders bacilli.

Finally a sporotrichum infection was thought of.

On 23 March at the suggestion of Chief Physician Dr.Tomasczewski serous secretion and tissue scraped from the flcorand from the edge of the ulcer were transferred to five tubesof Sabouraud's maltose agar. Six days later a colony of Sporo-trichum beurmanni began to grow uncontaminated in one tube;eight days after the transfer a sprotrichum colony also becomevisible in a second tube, alongside several diplococcus colonies.The rest of the tubes remained sterile.

On 31 March nine more maltose agar tubes were preparedwith the serous secretion of the ulcer. On 3 April sporotri-chum colonies began to grow in a pure state in all the tubes.

After another inoculation done on 4 April with the samekind of material numerous colonies of Sporotrichum beurmannibegan to grow in a pure state on the evening of 6 April, or inother words after only two days.

That the cultures at first grew so scantily and slowlyand later luxuriantly and increasingly faster must be attributedto the fact that a short time before the first inoculation ofthe secretion the ulcer had been sprinkled w.th iodoform powderand the fungus thereby temporarily inhibited in its d,velopment.

When the diagnosis of sporotrichosis appeared confirmedby the first culture, the serous secretion of the ulcer was re-peatedly examined directly under the microscope for fungus

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elements. .'or this purpose .... r Da .. rations wer-- made, fi..edin absolute alcohol, and stained in some cases by the ordinaryGram method and in others by the modification riven by Miuch.

In all these preparations, b .ides diplococci and strep-tococci there are to be found singly or in small groups ovalor irreg-ularly roundish or angýularly otitlinud dark violet, dif-fuse, grainy, or only marginally stai!,nd snore-like bodies(formes 71obuleu-es?) and also fine, li,-ht-refracting, slightlycurved or more or less strongly arched forms, here and thereputting out lateral branches shorte.- to varying extents, muchinferior in width to the size of the spore-like bodies and alsoto the cocci.

They show an irregular coloration, dark violet partsaltern, ,ing with pale blue, barely visible ones at more or lessregula, intervals. The stained parts constitute forms of vary-ing size and multifarious shape; sometimes they are round,sometimes oval, conical, or more angular in outline.

These threads are faintly reminiscent of Much's granularform of the tuberculosis bacillus, but without being identicalwith it; for repeated examinations of the smear preparations fortuberculosis bacilli with Ziehl-Neelsen staining came out nega-tive.

On the other hand the threads do coincide morphological-ly and tinctorially with the forms first found in tissue by '.Arndt, specificallyin abscess of the testicle and in necroticparts of testicles and lymph glands of sporotrichotic rats andwhich he interprets as mycelium threads of Sporotrichun beur-manni. (See also Figures 1 and 2 under experiments with ani-mals.7

I should like to call particular attention to this find-ing, because it has not been possible before to detect elementsof Sporotrichum beurmanni with certainty in man, either insmears of sporotrichotic material or in the tissue.

In further confirmation of the diagnosis I also repeated-ly examined my blood serum for its capacity both for spore ag-glutination and for complement fixqtion, keeping to the recom-mendations of F. Widal P. Abrami, L. Joltrain, Et. Brissaud,and A. Weill [see Note].

[Note] ",Lvcotic, etc., Serodiagnosis," Annales de l'In-stitut Pasteur (Annals of the Pasteur Institute), Vol 94, 1910,No 1.

The first examination was undertaken on 4 April, that isfour days after the specific therapy was begun, and at a time

when the clinical symptoms were still fully developed.The complement fixation experiment gave no results that

could be evaluated, as two control experiments also showed pro-

nounced deflection of complement. For the control experiments

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sera were used which were obta.. - fr: two f,:r'oas certainlyunaffected by mycotic disease, ox:e o: .."on -,ivan a posi-tive and the other a negative 7.,aoucrxxrnn reaction.

In setting up the experiment I deviated from the customof the French authors to the extent that I used not the wholeculture suspension, containing threads and conidia, but an ex-tract of it, in order to obtain as homogeneous an extract aspossible, and perhaps the source of error is to be sought inthis modification, assuming that the other components used inthe experiment did not fail.

The test of spore agglutination undertaken on the sameday gave a titre of 1:80. The spore suspension used was pre-pared from a culture six weeks old and not killed, and wasdiluted until it contained about 150 spores in the microscopicfield of vision with ocular 4, objective 6 (L. Leitz). The ag-glutination was observed under the microscope during the firsttwo hours after the start of the experiment. The agglutinatingeffect of the serum showed uD within the first quarter hourin the 1:20 dilution and at the end of the first hour in the1:40 dilution. By the end of the second hour a definite agglu-tination had taken place even in the 1:80 dilution; the sus-pension contained, besides i1olated elements, numerous clumps,varying in size, of spores sticking together. The controlserum, which came from a non-mycotic patient and showed no Was-sermann reaction, agglutinated the same spore suspension withinthe first two hours only in a dilutior of 1:?0, the agglutina-tion not manifesting itself until fi'e quarter-hours after thebeginning of the experiment.

In the pure spore emulsion not mixed with serum no traceof agglutination was found.

On 28 April both experiments were repeated. The ulcerat that time had been filled with epithelized granulations foreight days, and the other clinical symptoms had long sincedisappeared without a trace.

The complement fixation experiment, in which this timethe whole suspension from a three-week-old culture was used,containing threads and spores, gave a pronounced inhibitionand the control a complete solution of the sheep's blood cor-puscles. (The Wassermann test done on my blood the same dayby Mr. C. Hoffmann came out negative.)

On the other hand in two tests undertaken for the purposethe serum this time did not agglutinate the spores. For oneof these tests the epore suspension was prepared from cultureseight weeks old and not killed, for the other from cultures sixweeks old and formalinized.

On 1 May both experiments were finally run the thirdtime, and this time neither agglutination nor complementfixation took place.

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One thing that shows up from these :xscri:ments is thatthe serum still possessed the property of compiement deflectionat a time when it proved to be no longer capable of agglutina-tion.

Treatment and Further Course

When the diagnosis of sporotrichosis appear,-d confirmedby the starting of the first Cultu...s, I took 0.2 of calciumiodide internally three times on the first day, an-, because ofa severe iodide catarrh, 2 g of saiodin the next day and 2 andlater 3 g a day for 1 1/2 months thereafter. The ulcer wasonly dressed with a sterile bandage. During the very firstdays the swelling and painfulness of the regional lymph glandsand lymph vessels yielded. Only a few of the lymphangiticnodules could still be felt for a considerable time, about twoweeks.

The ulcer itself healed relatively slowly.

Photograph no. II shows the ulcer five days after begin-ning of the calcium iodide treatment. A tongue of granulationtissue extends into the ulcer from the edge. But along withthis sign of incipient healing a slight aggravation could be

GRAPIiC' i -7DUCIBLE

Photograph II. The same ulcer five days after be-ginning of the calcium iodide treatment, whosespecific effect is clearly brought out here. Atongue of granulation tissue extends into the ul-cer from the edge. In comparison with the firstpicture, at the same time, a greater depth of thefloor of the ulcer can be discerned; this becameevident after only a one-day interruption of thecalcium iodide treatment.

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discerned on the day the photo,--rr'h was t"aen; this manifesteditself in Creater depth of the floor and grc.ter serration ofthe edge, and set in after only a one-day interruition of thesaiodin treatment.

On 22 April 1910 the ulcer was completely filled withepithelized granulations. Under pressure, however, drops ofwhitish-yellow pus ooze"! out at several points from very small,ilmost imperceptible openings. This pus containced numerousGram-positive cocci, singly, in pairs, and grouped together inlittle chains or clumps, but no formations suggesting fungTuselements. Nor were any sporotrichum colonies obtained by inocu-lating it in Sabouraud maltose agar tubes, but only cocci colo-nies.

By the beginning of !ay the ulcer was completely healed.

Animal Experiments (on Two Nale Rats) with Material Obtainedfrom the Process Described Atove

Rat I

On 8 April a black and white rat was injected intraperi-toneally with three sporotrichum colonies from the size of apea to the size of a bean, two weeks old and in suspension ina physiological solution of common salt.

On 16 April the animal was again injected intraperitone-ally with a two-weeks-old culture.

Immediately after this inoculation the rat died, as wasfound in dissection, from intraabdominal bleeding. Very probab-ly the culture had been injected into the vena cava inferior orinto one of its branches, for in the later histological examina-tion of the lung, besides numerous gonidia, branched fungusfibers forming here and there a more or less dense myceliumnetwork were found in almost all the arteries of the lung in anabundance and an unambiguous form not observed in the organs ofany other experimental animal to date.

Although the first inoculation had occurred only eightdays before, considerable changes were already discernible upondissection.

The lymnh glands of the gantrohepatic omnntum, of thegastrocolic and gastrolienal ligarents, were swollen to the sizeof peas, and were of a hard consistency and smooth surface. Inthe gastrocolic ligament they formed conglomerates of varyingsize which were lying like a hood on the greater curvature ofthe stomach in the form of hard, lumpy tumors. When cut throughthey usually showed a gray-red, smooth, moist surface of sec-tion. One lymph gland was abscessed in the center; another con-tained in its center a cavity as large as the head of a pin andfilled with yellow, friable masses.

The other organs, in particular the testicles and epidi-

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dymes, show::u no macroscopically .xc. :ni'l~e -.'I.-*os.

Sabouraud maltose agar tubes wcre inocu',-ted with thetissue fluids of all the organs, with blood tarken from theheart, with the pus and the friable m(SS of the lymph glands,and with the urine. Un 20 April numerous sporotricnum coloniesstarted un in the tubes inoculated with blood from the heart,with tissue fluids from lung, liver, kidney, er., and fromthe globus major of the epididymis. On 21 April ,norotrichumcultures from the pus and the fri.ible na?-Lces of t', mesentericglands. The other tubes (testicles, Clobus minor of tne epi-didymis) remained sterile. A mold fungus developed in theurine test tube.

Smear preparations were made from the pus from the cavi-ties of the glands and the necrotic irTssec; they were fixed inabsolute alcohol and stained by Gram's i:ethod.

In the pus numerous sharply ',-fi-ed round, ovol, fusi-form, and more angular dark blue focms -;ere found, some of whichexhibited small round or conical bud-like appendages and a fewmore or less long, fine, or somewhat thicker, here and therebranched threads, in which stained and unstained parts alternat-ed almost regularly. The stained parts stand out sharply withtheir dark-blue color from the unstained, are of varying sizeand sometimes round, sometimes oval, sometimes angular shape.The pale parts of the threads are bounded by fine, pale blue,barely visible outlines; with contrast staining with safraninethey take on a dull red color.

These forms are found more beautifully and definitelydeveloped and also considerably .ore numerous in the smears fromthe friable masses. Nu.Lnerous usually single but sometimes pairedelongated bodies, slightly rounded or square at the ends,with diffuse violet staining, are also seen, and here and therelarger round formations of irregularly 1-rainy staining (seeFigure 2). Since sporotricha can be g-rown in pure culture fromthe friable masses and the pus, the above-described formationsmay be assumed to be elements of Soorotrichum beurmanni, i.e.mycelia and spores.

Parts of the lung, liver, and spleen, half a kidney, onetesticle, and several inflammationilly altered peritoneallymph glands were fixed and hardened in ascending percentagesof alcohol, embedded in paraffin, and cut in serial sections.These were stained by various methods (van Gieson, Pappenheim,Gram, elastic fiber staining).

The lung was found to be permcated with peribronchial,more or less extensive infiltrates made up of proliferatedconnective-tissue cells, mononuclear round cells, and numerousplasma cells of the Yarschalk6 type.

The livex shows only sligqht cm•ql-celled infiltration inthe immediate vicinity of the branchus of the portal vein.

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The p 5in c 'L c: . . . . . .,oh.,i:,,',is to be seen of the actual -l.nýLZ&<r t-zCue. in i"s piace isa diffuse infiltrationi c,:,.po:ud of ::o.uclear ro,;nd cells,epithelioid cells, nu:.;crous irrcf-ularly scattered .-iant cells,and scattered plasma cells, showli.; ni;.:-rous m~croscopic andsomewhat larcder alsceus for.atiors. The epithelioid cells insome places for-. tubercular nodulc';, comietiz-es conteining giantcells; they are surrounded by a zone consisting o, proliferatedconnective-tissue cells a-nd round cells.

The abscesses, which here and there possess a capsule ofconcentric layers of connective tissue, are lTrige1y composed ofwell-preserved leucocytes with lobate nuclei, though some ex-hibit necrotic portions of varying size, situated more towardthe center.

One of these necrotic portions in a fairly large abscesswhen Gram stained shows nunerous -cattered dark violet bodiesof irregular curvei outline, which ac'e for the mo=t part sur-rounded by a fairly wide pale aieola, whi'.e the central irregu-larly stained part possesses a crinulated apnearance.

We obviously have lere thv "for.es -lobuleuses" of theFrench authors, which are to be interpreteu as spores.

In the immediate vicinity of this necrotic spot was founda second, less extensive necrosis, which contained the above-described threadlike forms, probably corresponding to mycelia,in fairly abundant quantity (see Fijure 1).

Iat IIOn 8 April 0.2 g of the clear, serous secretion of the

ulcer was injected intraperitoneally into a white rat sixmonths old.

On 23 April a few drops of blood were taken from the ratunder sterile conditions and transferred to maltose agar Inthi inoculated tube yeast fungi and coccus colonies grew. outino sporotricha.

On 7 Xay 1910 the rat, apparently healthy, was bled todeath by opening the cai'otid under licg,.t alcohol narcssis.

Examination of the serut• for sporoa-glutination and forcomplement fixation turntl out negat-,v.

Dissection. -- In t'ie mesenterium of the large intestinethere were several moderately hard glands, from the size of alentil to the size of a bean, which showed a fgrayish red, moist,smooth surface of section.

No pathological changes could be observed macroscopical-ly in the other organs.

Blood and tissue fluid from 3,;n7, liver, !:idney, spleen,

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;esticle'.n, ed" ...... Z,• C•:'..,i~Ž a'e>':tc~r./,zC ":0rc inoculated onmaltose agar (3abouraud).

On 12 :,ay colonies of Thoro" l'rtnrr began togrow in a pure state in the tubu.s iu:u:.. wits blood andparenchymatous fluid from the cpiiidy.'. '.21- other tubes re-mained sterile.

Histological chan:ýr.; were : .... y i, the !un-; theycompletely corresponded to those iuD:hu irn t'h .u1 of rat I.

The normal structure can uc di:ocrrrIrd in t.e swollenmesenteric glands, but they con.ain, thuugh only here and there,Gram-positive formations of varying form, -- fira;tly, straightor more or less curved thread.;, sqzlýýre or slightly rounded atthe ends, which are stained intensely dark violet, either ir-regularly after the fashion of the threaIs already several timesdescribed (see Figure 4) or diffusely, and in the latter caseexhibiting shallow or deeper indenta.tions on the sides (seeFigure 3); secondly, square-oval, in.tensely stained formationssituated in pairs, which are similar to those found in thefriable lymph gland masses of rat I (see Fig-,ure 3); and lastly,thick rods tapered toward the ends, with pale ends and irregu-larly stained light to dark blue center, which are reminiscentof de Beurmann and Gou-erot's "faomes myceliennes courtes," ifnot in fact identical with them (see F'igure 5).

If all these forms, as perhaps we are entitled to assume,are elements of Sporotrichum beur7-.nni, they constitute a fur-ther illustration of the multiplicity of forms of this fungusin animal tissue.

With regard to the literature I refer the reader to thework of G. Arndt cited above, which contains in its appendix adetailed list of the works published thus far on sporotrichosis.

Explanation of the Figures

Fi-ure 1. -- i.ecrotic portion of an abscess of a mesen-teric gland of rat I. Gran-;'Welgert staining, pre-staining withlithium carmine. Zeiss ocular 4, oil immersion. From the redground, consisting for the most part of disintegrated cells,fine, more or less curved threads stand out, putting out shortlateral branches here and there; in their protoplasm dark violetstained, irregularly outlined parts alternate with pale parts atmore or less regular intervals.

These forms have already been found in animal tissue byG. Arndt and interpreted as myceliwn fibers of Snorotrichumbeurmanni.

Fit,_ure 2. -- Smear preparation from friable masses foundin the cavity of a mesenteric gland of rat I. Gram stainingwithout constrast staining. Zeiss ocular 4, oil immersion.

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The field of vision cc>t. - groot nua.ob•,r of the ir-regularly stained threads of vairyi.ng thickness just described,alongside intensely and diffusely dark violet stained, quiteirregularly shaped bodies sometioes occurrin,7 in pairs, andquite scattered larger round formations of irregularly grainystaining (spores?).

Fip-es 3. 4, and 5. -- Parts of an cnlar,--'e mesentericgland of rat II, which nevertheless still shows normal struc-ture. (Gram, stainin- with lithiu:n caruine.) They chow Gram-positive formations of varying shape, which correspond withgreat probability to elements of Soorotrichrm beurmanni. Figure3 in addition to a pair of the irregularly shaped bodies justdescribed (Fijure 2) contains a diffusely stained thread exhib-iting shallow indentations, Figure 4 a quite irrejularly stainedthreadlike form, and lastly FIgure 5 an irregularly stained rod,tapered at the ends, which is reminiscent of de Beurmann's"forme courte myc4lienne."

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