THE TREPONEMA PALLIDUN x' ORSERVATIOSS ON ITS OCCURRENCE AND DEXONSTRATION IN SYPHILITIC LESIONS* BENJAMIN WHITE AND OSWALD T. AVERY BBOOKLYYN, N. Y. The discovery by Schaudinn and Hoffmann' in 1905 of a spirillum in various luetic lesions marked the beginning of a decided advance in our knowledge of the etiology of syphilis. The announcement of the discovery, while most conservative in its assertions, gave a fresh im- petus to the study of the many phases presented in the parasitology and pathology of this disease. A wide-spread interest was awakened and many investigators directed their attention to the problems in this new field of research. From the results of many of their observations we have already gained a new conception of the cause, course and treatment of lues. The organism found by' Schaudinn and Hoffmann was first de- scribed by them under the name of Spiroch&a pallida, but a more intimate study of its morphology led them to forsake its classification under the spirochaete and to rename it the Treponema pallidu.m.t Much careful study has been given to the biologic nature of this organism, its demonstration and its behavior toward staining reagents. No less attention has been directed to observations on its occurrence in specific and non-specific lesions, and to the effect produced by both local and general treatment on its presence in such lesions. The evi- dence thus far available seems to show that this spirillum differs in its characteristics from all other known spirilla and thus probably con- stitutes a new species. Its absence from non-specific lesions and its almost, constant presence in specific lesions are being demonstrated repeatedly, while its general disappearance from these lesions under local and systemic treatment is an observation reported from many sides. These facts adduced from such evidence would seem to estab- lish this organism as the actual infecting agent in syphilis. Owing *From the Department of Bacteriology, Hoagland Laboratory, Brooklyn. 1. Schaudinn and Hoffmann: VorlYu5ger Bericht iiber das Vorkommen van Spirochreten in syphilitischen Rrankheitsprodukten und bei Papillomen. Arb. a. d. k. Gsndhteamte, 1905, xxii, 527.- Ueber &iroohceta. @.?idu bei Syphilis und die Unterschiede dieser Form gegentiber anderen Arten dieser Gattung. Be& klin. Wchnschr., 1905, No. 42, p. 673. 412 TEE TREPONEYA P;LLLIDUhf to the fact, however, that the Treponema fulfils only one of the con- ditions of Koch's law-since, so far, all attempts to cultivate it have proved futile-we can make no definite statement concerning the speci- ficity of its Se. Although this further confirmation is lacking, yet in the clinics and among the private practitioners in Europe, and to a small but increasing extent in this country, the presence of the Treponema in a suspected lesion is held as establishing the specific nature of the in- fection, and therefore its demonstration in such cases is considered as a valuable diagnostic aid. With the older methods of observation, on the other hand, often a delay of several weeks was necessary, de- pending on the appearance of secondary symptoms, before a `definite diagnosis could be made. An early diagnosis is now asserted to be of distinct advantage in the treatment of this disease. I.-THE DE;MONSTRATION OF THE TREPONEMA The inaccessibility of much of the literature, combined with the more or less unsatisfactory results obtained with the intricate and time-consuming staining methods, may account for the lack of initia- tive on the part of many of the medical men of this country to avail themselves of this aid to diagnosis. It is therefore the purpose of this paper to give a critical review of the various methods recommend- ed for staining the Treponema, to describe a simple and reliable proce- dure for its demonstration, and, further, to report a series of observa- tions made in about one hundred cases, both syphilitic and non- syphilitic in character. As a diagnostic measure, the recovery and demonstration of the Treponemu pallidurn from primary lesions is probably of greatest im- portance, and in this instance the attempt usually meets with a greater degree of success than is the case when later lesions are examined. Its presence in some of the secondary lesions is frequently less con- stant and its demonstration presents greater difficulties. Reports of positive findings in the pustules and ulcers of the tertiary stage are comparatively few in number. The recovery of the spirillum from the circulating blood in primary and secondary syphilis has been reported, but the nature of the technic renders any description unnecessary here. The untreated chancres, the condylomata, the mucous patches and moist papules offer the most promising field for investigation, but the precise nature of the material to be obtained from these lesions is of primary importance. Many failures to demonstrate the Treponema are traceable to the faulty manner of preparing the specimen rather than BENJAMIN WHITE-OBWALD T. AVERY 413 to the method of staining employed. Some authors advise that a small incision be made in the lesion with a lance or a Hagedorn needle, and the resulting drop of blood spread in a thin film on a cover glass or slide. This method is not always feasible or satisfactory. The method used in the present resea.rch is simple in execution and produces uniform and dependable results. Since this step constitutes so important a factor in making a successful preparation its detailed description may be justified here: The lesion is washed when necessary, then thoroughly cleansed by wiping with gauze. The juncture of the necrotic with the sound tissue, and also a part of the floor of the sore, are then curetted with a small curette (the chalazion eye curette answers the purpose admirably), or scraped with a scalpel until the detritus and superficial tissue are removed and a slight flow of blood is produced. The lesion is then sponged with dry gauze until the blood has ceased to flow and clear serum is seen to ooze. A. drop of this serum is then spread on a per- fectly clean glass slide in the thinnest possible uniform film. The preparation, after being allowed to dry in the air, may be fixed either by carefully passing through the flame three times, by immersion in ethyl or methyl alcohol, by osmic acid or in the osmium tube of Hamm. ' The inability of early investigators to demonstrate- the existence of the Treponema in syphilitic lesions may be ascribed in part to the inadequacy of the optical appliances then available and to the dif?l- culties presented in impregnating its cell substance with any of the usual staining reagents. The perfecting of apparatus for the pro- duction of dark-ground illumination has made it possible to render these poorly refracting cell bodies visible, and to enable one to observe them in a living condition. This method of direct examination is by far the most satisfactory, not only for making a rapid diagnosis of suspected material, but for an intimate study of the morphology and manner of reproduction of these organisms. The employment of this method is, however, limited by the nature of the apparatus required.* The simplest, and for general purposes, the most satisfactory-method of demonstrating the Treponema is, therefore, by means of some ap- propriate staining procedure. Such a vast array of staining methods has been advocated that a search for one which involves no elaborate technic, which is rapid in execution and which gives satisfactory pre- parations leads only to bewilderment. A great majority of these 2. Eecently this method has been, ,described . in detail by Harris and Corbus: The clinical value of the spirochseta; pallida ia the diagnosis and treat- ment of syphilis. Jour. Am. Med. Awn., 1908, li, 1928. -114 TEE TREPONEMA PALLIDUdi methods, when a.ttempted by one not skilled in laboratory manipula- tions, yield discouraging results, and this means ,of diagnosis is then abandoned or intrusted to those who have given more or less study to the technic involved. The difi?culty in demonstrating the syphilis organism by any of the usual laboratory methods is largely due to the fact that the protoplasm of the Treponema exhibits only a slight affinity `for the majority of the anilin stains. Schaudinn and Hoffmannl were the fifst to succeed in producing stained preparations of these spirilla. They found that in smears, fixed with osmic acid and allowed to remain immersed in Giemsa solution for twenty-four hours, the Treponema acquired a deli- cate rose color, while other spirilla which might be confused with the Treponema were more intensely stained. Since the publication of this method, the Giemsa solution, particularly the preparation of Griibler, has been widely employed for this purpose. Many modifications in its use have been advocated, each being supported by claims as possessing distinct advantages over the original procedure. For the most part these variations yield no more satisfactory results than the method of Schaudinn and Hoffmann. One modification, however, that de- scribed by Schereschewsky,g is so simple in detail and produces such excellent results that it has been almost exclusively employed in the present investigation. This method will be fully described in another part of this paper. When it was learned that a demonstrable micro-organism was pres- ent in the lesions of syphilis many bacteriologists resorted to the com- mon staining reagents in the hope of discovering a method which would render the use of the more delicate- eosin and azure solutions unnecessary. Methylene blue, either in watery or alcoholic solution or prepared according to the method of Loeffler, has been employed*by Barrel and Burnet,' Weitlaner" and others who have found that it renders the Treponema visible and imparts to it a distinct blue color. Others, among them, Bandi and Simonelli,6 Ehrlich and Lenartowicz' 3. Schercschewsky : Daa Verhalten der Bpirooh&a pallida ( Schaudinn ) bei der Giemsaflrburg. Centralbl. f. Bakteriol., 1908, No. 45, p. 91. 4. Borrel and Burnet: Pro&de de diagnostic rapide dea lesions syphil- itiques, Compt. rend. Sot. de biol., 1906, No. 49, 212. 5. Weitlaner : Noch einiges tiber Bpirochceta paallidu. Klin. thcrap. Wchnschr., Vienna, 1905, No. 12, p. 1124. 3. Bandi and Simonelli: Ueber die Anwesenheit der fipirochtxta pallida in sekundiir-syphilitischen Manifestationen und tiber die zu ihrem Nachweis angewendeten Wrbungsmethoden. Mtinchen. med. Wchnschr., 1905, No. 52, 1668. 7. Ehrlich and Lenartowicz: Ueber Fsrbungen der gpirochceta pallida fur diagnostische Zwecke. Wien. med. Wchnschr., 1908, No. 58, p. 1018. BENJAiUIN WHITE-OBWALD T. AVERY 415 hare obtained satisfactory results with Ziehl's solution of carbol fuch- sin, while Gonder and Hoffmann, Ploeger,* Herxheimer,O Oppenheimer and Sachs,`O and Scholtzll advocate the use of gentian violet in a so- lution of anilin water, or better in dilute carbolic acid. The intensity of the stain produced by the various anilins seems to be markedly in- creased by the use of phenol, either in dilute solution as a solvent for the stain or in combination with other substances as a mordant. Proca and Vasilescu12 first mordant the preparation with a solution of tannic acid in 5 per cent. carbolic acid and then stain with carbol gentian violet. Flexne+ has reported favorable results obtained with this method. Quite different from any of the above methods is that described by Stern." The air-dried film is fixed by heating at a temperature of 3'7.5 C. for several hours. The slide is then immersed in a colorless glass vessel containing a 10 per cent. watery solution of silver nitrate. The vessel is allowed tb stand in diffuse daylight until the film appears brown with a metallic luster. If the action of the light has not been too intense, after thoroughly washing in water, the Treponema should appear evenly stained a deep brown with little precipitate on the slide. Flexner18 advises that the reduction be allowed to take place slowly & weakly diffuse light, as otherwise the protoplasm appears coarse and the contour is more or less broken and uneven. With this stain the cell body is somewhat swollen and does not show the delicate appear- ance produced by eosin-azure stains. This has li'ttle significance from a diagnostic standpoint, as none of the other characteristics are affected. This method has been repeatedly tried in this laboratory and has given positive results when it was impossible to demonstrate the presence of the Treponema with many of the other stains. The Treponema im- pregnated by the above method is shown. in b-b in the accompanying illustration. 8. Ploeger: Die SpirochKten bei Syphilis. Mtinchen. med. Wchnschr., lQq5, No. 62, p. 1381. 9. Herxheimer : Zur Kenntnis der rSpiro&u%a pallida. Mlinchen. med. Wchnschr., 1905, No. 52, p. 1861. 10. Oppenheim and Sachs: Eine einfache und schnelle Yethode zur deut- lichen Darstellung der Npirochreta pallida. Deutsch. nied. Wchnschr., 1905, No. 31, p. 1156. 11. Scholz: Ueber den Spiroch%tennachweis bei Syphilis. Deutsch. med. Wchnschr., 1905, No. 31, p. 1467. 12. Proca and Vasilescu: Sur un pro&l6 de coloration rapide du Npiroch@ta padlida. Compt rend. Sot. de biol., 1906, No, 57, p. lo+.. 13. Flexner: Npirochceta (Treponema) pallida and syphilis. Jour. Exper. lied., 1907, No. 9, p. 404. 14. Stern: Ueber den Nachweis der &koch&a paEEidcc im Ausstrich mittelst der Siibermethode. Barl. klin. Wchnschr., 1907, No. 44, p. 400. 416 THE TREPONEMA PALLIDUM In this laboratory all of the above mentioned methods, and further those advocated by Goldhorn,`" Wood, Hastings,1o and Gradlel? have been thoroughly tried out on material known to contain the Treponema in large numbers. In addition to the testing of these methods original attempts were also made to stain this organism with certain anilin colors, among them thionin, pyronin, safrinin and the three-solution diptheria stain of Neisser. To summarize briefly the present observa- tions made on the respective merits of these various methods it may be stated that : (a) of the simple anilin colors, fuchsin, methylene blue and gentian violet, when they do not yield entirely negative results, impregnate the protoplasm of the Treponema only to a slight degree; (b) the action of these anilins is intensified by the -use of phenol or tannin as mordants, but at the best their employment leads to unsatis- factory preparations ; (c) the silver impregnation method of Stern is thoroughly dependable, but the length of time required for its execu- tion constitutes more or less of a disadvantage; (d) the Treponema is stained more intensely by solutions containing eosin and azure; (e) the Giemsa solution aa used by Schaudinn and Hoffmann, and in many of its later modifications, may usually be depended on to produce well stained preparations, but with its employment are associated certain unsatisfactory features in the matter of time consumption, etc. The results obtained by the method as modified by Schereschewsky have proven uniformly satisfactory. This fact, combined with its rapid- ity of execution and simplicity of technic, has led to its adoption in preference to all others for the purposes of the present investigation. The technic in detail is as follows: The smears are obtained in the manner already described and after being allowed to dry in the air are carefully passed through the flame three times. The staining mixture is freshly prepared by adding thir- teen drops. (from a dropping bottle) of Giemsa solution (Griibler) to 10 C.C. of a 0.5 per cent. watery glycerin solution. The mixture is then heated to boiling, immediately poured on the slide and allowed to re- main for three to five minutes. The stain is then poured off and the slide washed with neutral distilled water. The slide is dried by rapidly shaking it in the air and a second application is made for the same length of time. As a rule two applications suffice to impart to the 15. Goldhorn: A rapid and certain method of staining Spiro&&a pallida. Proc. New York Path. Sot., 1905, No. 6, 169. 16. This stain has recently been recommended by Geraghty: Johns Hopkins Hosp. Bull., 1908, XIX, 364. 17. Gradle (H. S.): A clinical stain for the Spiro&&a pallida. Jour. Bm. Med. Sssn., 1908, 1, 1265. BENJAiUIN WHITE-OBWALD T. AVERY 417 smear a distinct pinkish tinge. Should the pink be too faint a third application is made. When the desired shade is reached the slide is washed as above and dried by shaking or with fine blotting paper. The preparation is then examined with a one-twelfth oil-immersion lens. In order to obtain ideal results certain precautions must be heeded. All vessels with which the stain comes in contact must be perfectly clean. Before mixing each fresh lot of stain the test tube used Bhdd be cleansed by scrubbing with clean cotton and alcohol, then rinsed with distilled water. Any deposit of stain about the neck and lip of the dropping bot'tle should be removed by carefully wiping with filter paper. The water used in making the glycerin solution and for wash- ing the Blide should be neutral, as the least degree of acidity causes the formation of a precipitate. Should any of these precautions be neglected it will be found that a heavy bluish precipitate forms in the mixture on boiling and its staining ability is thus impaired. The ex- act tint of the smear to be attained is a matter of no great importance and is easily determined. After a few trials in which the importance of the above details is realized one may expect to produce satisfactory preparatione. In the preparations thus stained the Treponema should appear a deep pink with the background pale in comparison. Fixation by heat has a tendency to straighten the convolutions, but this may be easily overcome with the exercise of a little care. The Schereschewsky stain, on account of the glycerin it contains, produces a slight swelling of the cell body. With this stain, however, a sticiently accurate exposi- tion of the morphologic features of the Treponema is produced. The Treponema pallidurn is a delicate spirillum varying in length from 4 to 14 microns, and having a breadth which is generally less than 0.25 microns. The ends are somewhat pointed The convolutions, accord- ing to HofEnann and Halle, lo have a width of 1 to 1.2 microns, a depth of 1 to 1.5 microns, and are usually 6 to 14 in number, although at times longer forms are Been which exhibit as many aB 20 to 24 convo- lutions. The angularity and the regularity of these windings are mark- edly characteristic of this particular organism. A careful microscopic examination renders the differentiation of the Treponema from all other Bpirilla certain. The majority of spirochetes, on account of their coarBer form, their irregular and broader convolutions and their greater aRinity for stains, can scarcely be confused with the syphilis spirochete. The flppirochceta refringens, frequently found in suspicious lesions, is 18. Hoffman and Halle: Ueber eine bessere Darstellungsart der &~~rocluetu pallida im Bus&rich. MUnchen med. Wchnschr., 1906, NO. 63, p. 1518. 418 TEE TREPONEMA PALLIDUY easily &BtiUgUiShed by its greater width and its more wavy form. Should doubt arise concerning the specific nature of the organism under examination the material should be examined in the fresh state best by means of the dark field illumination. When this is impossible the preparation may be fixed rapidly with osmic acid and then BtaiUed for several hours-preferably twenty-four-with a mixture containing one drop of the Giemsa solution in 10 C.C. of neutral distilled water. In all of the examinations made in this laboratory no di5culty has been experienced in establishing a diagnosis by means of the Schereschewsky method. II.-THE OCCURRENCE OF THE TEEPONEMA IN SY+HILITIC LESIONS I8 It seems unnecessary to review here the large number of reported observations on the occurrence of the Treponema. in syphilitic lesions. The results of these observations may be briefly summarized thus: 1. The Treponema has been found in the great majority of untreat- ed primary and secondary lesions. Negative fiUdiUgB in such specific lesions are probably attributable to faulty technic. 2. The examination of material from tertiary lesions usually yields negative results. A few positive findings have been reported, however. 3. The Treponema has not been found in non-specific lesions. 4. Both local and general mercurial treatment tend first to make these spirochetes degenerate and later to cause their disappearance from the lesion. With a view toward gaining further evidence in this direction a series of observations on 101 cases were made extending over a period of one year. A certain routine procedure was established which con- BiBted in Obtaining the material from the various kBiOUB direCtly upon the admission of the patient to the clinic.20 In many instances subse- quent smears were made in order to verify the initial findings and with- out exception these were substantiated. Before publishing the results of these observations the appearance of the BeCOUdaq symptoms was awaited-when the examination was made on the primary lesion-so 19. A review of the literature extending through the year 1900 may be found in Kolle-Wassermnnn's Handbuch der pathogenen Organismen, Supple- mentary Volume I, 544; also in Mutter: Arch. f. Dermat. u. Syph., 1906, Ixxix. 20. We wish to express here our sincere thanks to Doctors Pedersen, States and other members of the staff of the Genito-Urinary Clinic of the House of Relief, New York City, and to Doctor6 Morton, Read and others of t.he Genito-Urinary Clinic of the Polhemus Memorial Clinic, Brooklyn, N. Y., for their many courtesies and hearty cooperation. BENJAMIN WHITE-OBWALD T. AVERY 419 that no doubt might exist concerning the specific nature of the case. These results may perhaps be best exhibited in tabular form. The findings in the cases of untreated primary lesions are con- firmatory of the results of others regarding the presence of the Trepon- ema solely in specific lesions. Local treatment with the various prepara- tions of mercury, such as calomel powder, blue ointment and black- FIXDINOS OF TEE !MZONEYA PALLIDW IN SPECIFIC AND NON-SPECIFIC LESIONS* Nature of Lesion. Primary lesions, untreated . . . . . . . . . Primary lesions, local treatment . . . . . Primary lesions, general treatment . . . . . Secondary lesions, untreated- M*cular syphiloderms . . . . . . . . . . . Maculopapular syphiloderms . , . . . . . . Condylomata . . . . . . . . . . . . . . . . Mucous patches . . . . . . . . . . . . Moist papules . . . . . . . . . . . . . . . Secondary lesions, treated- 33 13 1 1 1 3 1 3 3 2 I3 1 3 30 33 7 0 0 1 3 1 3 1 0 3 1 0 0 0 6 1 1 0 0 0 0 2 -2 3 0 3 30 100 53 00 00 100 100 100 100 33 00 50 loo 00 00 Maculopapular syphiloderms . . . . . . . . Puwtular syphiloderms . . . . . . _ . . . Mucous patches . . . . . . : . . . . . . Moist papules . . . . . . . . . . . Tertiary lesions. . . . . . . . . . . . . . . Non-specific lesions. . . . . . . . . . . - o These results agree closely with the observations of Harris and Corbus (Jour. Am. Med. Assn., 1908, li, 1928), which h&e appeared since the com- pletion of this paper and also with those of Geraghty (Johns Hopkins Hosp. Bull., 1908, xix, 384). 4 $a s-la SjEz 4 =I -- wash, may cause a disappearance of the Treponema from the supeticial tissues, although it undoubtedly persists for a time in the deeper lym- phatics of the lesions. Preparations made from mucous patches and moist papules, with the exception of three cases affected by treatment, were found to be unusually rich in spirochetes. Some of the prepara- 420 THE TREPONEYA PALLIDUY tions were taken directly from the surface of the lesion without pre- vious curetting. This substantiates the earlier view of the highly in- fective nature of these particular lesions. The greater proportion of negative results in the secondary lesions after general treatment as compared to those before this treatment is indicative of the effects of the systemic administration of mercury on the presence of the Treponema. The absence of the Treponema in the cases of tertiary syphilis, while being in agreement with previous findings and with the usually accepted idea concerning the non-infective nature of these lesions, might be explained by some fault in our present method of examination. Treponema pallidurn, under magnification X 1200; a-a were stained with the Scherescheweky stain; b-b with the Stern silver strin. From the review of the staining methods above recorded and from the observations made in the bacteriologic examination of syphilitic and non-syphilitic lesions the following conclusions may be reached: 1. The staining method as described by Schereschewsky appears to be the most satisfactory one for general use. 2. The presence of the Treponema palL&n in a suspected lesion may undoubtedly be considered as establishing the specific nature of the infection. 3. A negative result does not necessarily exclude the presence of syphilis. In this event further examinations should be made, and, when these lead to negative results a proper period of time should be allowed BENJAUIN ?VH?TE-ONVALD T. AVERY 421 to elapse in order that the appearance of the secondary symptoms may establish the diagnosis. In the clinic of the House of Relief the finding of the Treponemn has been considered as affording a positive diagnosis. The treatment when begun immediately on the report of a positive bacteriologic diag- nosis has led to most gratifying results.