896 EARLY SYPHILITIC HEPATITIS strains, with the exception that 2 of the 3 resistant isolates were fully sensitive to minocycline. Similar observati...

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strains, with the exception that 2 of the 3 resistant isolates were fully sensitive to minocycline. Similar observations have been made with staphylococci, in that some T.R. strains of Staph. aureus are sensitive to minocycline."3 In performing antibiotic and sulphonamide sensitivity tests, it has long been known that an excessive inoculum must be avoided, lest a drug-sensitive bacterium appear resistant. This is especially important in sulphonamidesensitivity tests. It is also important in testing the sensitivity of H. influenzce to ampicillin. In the presence of ampicillin, L-forms are produced by antibiotic-sensitive strains14 and an excessive inoculum may result in a false reading of ampicillin resistance. This applies to disc-diffusion, tube-dilution, and plate-titration tests. 14 11 We agree that a moderate and standardised inoculum is advisable and believe that in plate-titration tests with haemophilus an inoculum of about 104 viable units is suitable. Peak serum levels of tetracycline are usually 2-4 jig per ml after the normal oral dose (in adults) of 250 mg 6-hourly; after 500 mg 6-hourly, levels of 4-5 µg per ml are reached.’ 16 Higher levels again can be attained by parenteral administration. However, the degrees of tetracycline-resistance encountered here were so great as to preclude successful treatment with this antibiotic. At present, haemophilus meningitis is usually treated with either ampicillin or chloramphenicol. However, strains of H. influenzæ type b highly resistant to ampicillin have recently been encountered in the United States, Britain, and Germany.17-19 Tetracyclines have been used with success in the treatment of bacterial meningitis, including cases of haemophilus meningitis, either alone2O-22 or in combination with other antibacterial drugs.23 In view of the, present findings and because of its superior penetration into cerebrospinal fluid, chloramphenicol would seem to be a more suitable alternative to ampicillin in the therapy of this and other serious hsemophilus infections. The haemophili were isolated by Mr Malcolm Gibbs. I also thank the pxdiatric staff of the Adelaide Children’s Hospital for allowing me to use the case-notes of their patients. It is a pleasure to acknowledge the assistance of those pharmaceutical firms--especially Pfizer, Lederle, and Hoechst-which supplied the samples of pure antibiotics used. REFERENCES 1. Garrod, L. P., O’Grady, F. Antibiotic and Chemotherapy; p. 150. Edinburgh, 1971. 2. Williams, J. D., Andrews, J. Br. med. J. 1974, i, 134. 3. Hansman, D., Pidgeon, M. ibid. 1971, ii, 467. 4. Rogers, K. B., Zinnemann, K., Foster, W. P. J. clin. Path. 1960, 13, 519. 5. Hansman, D. Med. J. Aust. 1974, i, 715. 6. Annear, D. I., Norcott, T. C., Ruhen, R. B. Pathology, 1974, 6, 45. 7. Gots, J. S. Science. 1945, 102, 309. 8. Turk, D. C., May, J. R. Hæmophilus influenza;; p. 64. London, 1967. 9. Schneerson, R., Robbins, J. B., Parke, J. C. in Hæmophilus influenzæ (edited by S. H. W. Sell and D. T. Karzon); p. 13. Nashville, 1973. 10. Sinclair, S. E.J.Am. med. Ass. 1941, 117, 170. 11. Andrew, J. D., Tandon, O. P., Turk, D. C. Br. med. J. 1968, iii, 524. 12. Goldstein, E., Daly, A. K., Seamans, C. Ann. intern. Med. 1967, 66, 35 13. Mitchell, A. A. B. Br. med. J. 1974, i, 576. 14. Roberts, D. E., Ingold, A., Want, S. V., May, J. R. J. clin. Path. 1974, 27, 560. 15. McLinn, S. E., Nelson, J. D., Haltalin, K. C. Pediatrics, Springfield. 1970,

45, 827. 16. Kucers, A. The Use of Antibiotics; p. 279. London, 1972. 17. Thomas, W. J., McReynolds, J. W., Mock, C. R., Bailey, D. W. Lancet, 1974, i, 313. 18. Clymo, A. B., Harper, I. A. ibid. p. 453. 19. Medeiros, A. A., O’Brien, T. F. ibid. 1975, i, 716. 20. Drake, M. E., Bradley, J. E., Imburg, J., McCrumb Jr. F. R., Woodward, T. E. J. Am. med. Ass. 1950, 142, 463. 21. Hoyne, A. L., Riff, E. R. J. Pediat. 1951, 39, 151. 22. Lepper, M. H., Wehrle, P. F., Blatt, N. Am. J. Dis. Child. 1952, 83, 763. 23. Koch, R., Carson, M. J. J. Pediat. 1955, 46, 18.

J. FEHÉR 3rd Department

of Medicine, Semmelweis Medical

University, Budapest, Hungary MARGIT TIMMER


1st Department of Dermatology, Kallai Eva Hospital,

Budapest, Hungary L.


Department of Pathology, National Institute of Traumatology, Budapest, Hungary 17 out of 175

of early syphilis had clinical, biochemical, and immunological evidence of liver damage. Before penicillin therapy the histological appearance of the liver was abnormal in 14 of the 15 patients from whom biopsy specimens were obtained. In 7 cases, treponemes were seen in the liver. After two months’ penicillin therapy the extent and severity of the histological abnormality was reduced. In the repeat liver-biopsy specimens obtained after penicillin treatment no treponemes could be demonstrated. It is suggested that the hepatitis found in these 17 cases of early syphilis was produced by treponemes.



Introduction LIVER disease is often found in association with congenital syphilis and it also occurs in the late stages of the disease as fibrosis, gumma, and hepar lobatum. However, the association of liver disease with secondary syphilis in the early latent stages has not been well documented. A review by Hahncited pathological observations in only 9 cases. None of these was a clear example of early syphilis involving the liver. All the patients had died from causes other than syphilis or hepatitis. 8 of them had received arsphenamine before death and the remaining patient showed multiple hepatic granulomas, probably representing a later stage of syphilis. There have been few subsequently published case-reports of hepatic involvement in early syphilis.2-’ In some of these reports it was suggested that treponemes in the liver were responsible for the liver damage, but they were not demonstrated in the hepatic tissue of any of these cases. We have observed clinical and biochemical findings indicative of liver damage in many of our patients with early syphilis. These findings became normal after adequate treatment with penicillin. This observation and previous reports led us to investigate the immunological changes and the evidence of liver damage in patients with early syphilis and abnormal liver-function tests.

Materials and Methods In

1974, 175 patients with early untreated syphilis



dermatological department of Kallai Eva Hoswere 17 patients in whom liver-function them pital. Among tests indicated hepatic damage. Alcoholic patients and those treated before admission were not investigated. The diagnosis of syphilis was established by serological tests (v.D.R.L., Kolmer), and by demonstrating treponemes in local lesions. to


These examinations were carried out within six to seventeen weeks of infection. Serum-bilirubin, serum-glutamic-oxaloacetic-transaminase (S.G.O.T.), bromsulphthalein (B.s.p.) reten-


tion, thymol turbidity, gold sol, prothrombin-time, and alkaline phosphatase activity were used as liver-function tests.


Serum-protein was measured by the biuret reaction, and qualianalysis of proteins was carried out by paper electrophoresis. Serum IgA, IgM, caeruloplasmin, cx2-macroglobulin, tative

,C-globulin, and transferrin concentrations



by Mancini’s radial immunodiffusion.8 Sera from 25 healthy

people were used as controls. Sera were examined for antinuclear factor by the Hyland test. Hepatitis-B antigen (HBAg) was sought by a micro complement-fixation test. Blood-samples were obtained the day before liver needle biopsy. Liver biopsy was performed with a Menghini needle in 15 patients who had clinical and/or biochemical evidence of liver disease. Liver biopsy was contraindicated in 2 cases. The day after biopsy, penicillin treatment was started. Three to seven days after two months’ adequate treatment the biochemical and immunological tests were repeated, and in 7 patients a further liver-biopsy specimen was obtained after treatment. Liver-biopsy material for histological and histochemical examination was fixed in 6% neutral formol. After adequate fixation normal paraffin embedding methods were used. Serial 6um sections were cut. The following stains were used: hasmand eosin. Mallory’s trichrome, Van Gieson, Gomori’s silver impregnation, prussian-blue reaction for iron, Jobba’s bile staining method, and periodic-acid/Schiff (P.A.S.) reaction for glycogen. In all the cases Warthin-Starry’s stain for treponemes9 was also used. In 6 cases (4 before treatment, 2 after treatment with penicillin) the liver-biopsy material was prepared for electron microscopy. Blocks of tissue (lmm3) were immersed in cold buffered 2% osmium tetroxide and fixed at 4°C for an hour. The blocks were dehydrated in alcohol and embedded in ’Araldite’. Semi-thin and ultra-thin sections were cut on a Reichert OM U2 ultramicrotome. Semi-thin sections were stained with toluidine blue, the ultra-thin sections were contrast stained with uranyl acetate and lead citrate. Sections were viewed and pho-


tographed with a Tesla 242 BS electron microscope. The t test used for statistical analysis.



Clinical and Immulogical Observations TABLE I-DATA ON


’OccaslOnally. ,


= Annnuclear factor.


There was no significant difference attributable to either the age or the sex of the subjects (table I). Patients were aged between twenty and sixty-eight years (mean forty-four). None had a history of hepatic disease. All 17 patients had hepatomegaly; 4 of them also had splenomegaly. Routine laboratory data indicated hepatic dysfunction. The colloid lability tests seemed to be the most sensitive; however, S.G.O.T. activity was increased in several cases, while serum-bilirubin, prothrombin, and B.s.P. retention were abnormal in several cases. Serum IgG and IgM concentrations were significantly higher in the patients than in the controls (tablesand n). Serum-IgA concentrations were not significantly different in patients and controls. The increase in serum-caeruloplasmin was highly significant, while serum-transferrin concentration was significantly decreased. Cl:2-macroglobulin and &bgr;IC-globulin were not significantly altered.

Morphology A liver-biopsy specimen from 1 case showed no histological abnormality, either before starting treatment or after therapy. In the other 14 cases histological examinations showed varying degrees of abnormality. In the cases with minimal change there was proliferation of sinus endothelial cells and Kupffer cells, together with many leucocytes, eosinophils, and lymphocytes in the sinusoids. The periportal region was swollen, and the walls of the arteries and portal vein branches were thickened and infiltrated with inflammatory cells. The infiltrate often included neutrophils as well as mononuclear


cells, while in

some cases the vessel lumen was narrowed. These changes were found in 2 cases, and in 1 of them the walls of some arterioles were also infiltrated with fibrin. In these cases the architecture of the liver was not altered and there was no evidence of cholestasis. There was focal liver necrosis in the other 9 cases. Necrotic lesions sometimes involved only one or two liver cells, in other cases they were more extensive. In the nec-

rotic areas, neutrophil and eosinophil granulocytes, lymphocytes, and mastocytes were found and the recticulin structure was destroyed (fig. 1). In some areas of necrosis, fibroblasts and epithelioid cells were seen as well as an acute inflammatory infiltrate. Necrotic foci were found in all parts of the lobules, but they were more common in the periportal region and around the central vein (fig. 2). The necrotic inflammatory foci around the central vein appear to be characteristic of syphilitic hepatitis, being present in all our cases with liver damage. The walls of the branches of the central vein were thickened, with increases in reticulin and collagen fibres. Centrilobular glycogen content seemed to be reduced, although it was normal at the periphery. Glycogen content was markedly reduced in the necrotic

areas and in their immediate neighbourhood. Haemosiderin accumulation was found in one case, and intrahepatic cholestasis was observed in another case by light microscopy. Liver steatosis, Mallory’s bodies, or other tissue changes indicating alcoholic liver damage were not seen. In 7 cases treponemes were demonstrated in the liver-biopsy material. They were found in the inflammatory necrotic foci, in the sinus endothelial cells, in the Disse spaces, and sometimes in the intrahepatic bile capillaries (fig. 3). After two months’ antibiotic treatment no treponemes were found in repeat liver-biopsy material from the 7 cases in which treponemes were seen in the liver before treatment. The necrotic foci, except in 1 case, had disappeared ; there was no inflammatory reaction in the wall of the central vein; periportal connective tissue inflammation and arteriolitis were absent. In 1 case, however, the portobiliary connective tissue was extensively infiltrated with lymphocytes and plasma-cells, and some necrotic changes involving one or two liver cells could be observed in the liver parenchyma. It is noteworthy that this was one of the cases which showed abnormal immunological and laboratory values at the repeat examination. In all cases after therapy, collegen had been deposited in the sinusoids, in some cases collagen fibres appeared among the liver cells. In some cases the wall of the central vein was thickened with an accumulation of collagen and reticulin fibres. Treponemes were

Fig. I-Light micrograph of liver tissue from patient with early syphilis showing focal inflammatory reaction.

Fig. 3-Light micrograph showing treponeme.

Hasmatoxylin and eosin. Reduced to3 from

Wharthin-Starry (xlOOO).


Fig. 2-Light micrograph of liver tissue from patient with early syphilis showing lymphocytic infiltration of region around vena centralis.

Haematoxylin and eosin.


to3 from


of liver tissue from

patient with early syphilis

Fig. 4-Electron micrograph of liver cell showing tubularisation of endoplasmic reticulum and intracellular treponeme. (x 9200 i


demonstrated by electron microscopy in the


material taken from two of our cases before treatment. In both cases treponemes were found intracellularly and in the sinus endothelial cells. No treponemes were found in liver-biopsy specimens taken after treatment. Electron microscopy, in accord with the light microscopic observations, showed reduced glycogen content. The mitochondrial matrix contained abundant electrondense material. Lysosomes were increased in size and number. The endoplasmic reticulum was generally degranulated and tubularised (fig 4). Lipofuscin was seen in all the liver cells, and bile droplets were found in some cells. There was intracellular cedema in liver cells and in sinus endothelial cells. These cells were sometimes vacuolated, and lipid vacuoles were seen occasionally. In the specimens examined after penicillin treatment, ultrastructural changes were slight in the liver cells. Lipofuscin was increased and glycogen was decreased. Electron microscopy confirmed our light microscopic observations-i.e., that the healing of the syphilitic hepatitis resulted in an accumulation of collagen fibres both in the wall of the sinusoids and amongst the liver cells.

in 7 cases showed healing of liver damage and no treponemes. Syphilitic hepatitis healed with the accumulation of collagen in the walls of sinusoids and in the spaces between the liver cells.


We attach special importance to our case in which liver function remained impaired and serum-immunoglobins were abnormal after 2 months’ penicillin therapy. In this case piecemeal necrosis of the liver, lymphoid cell infiltration, and fibroblast proliferation were found. It is suggested that in this case the hepatitis of treponemal origin, in common with some cases of viral hepatitis, had induced an autoimmune hepatitis. This suggestion is supported by the finding of antinuclear factor in 1 of our cases, and the occurrence of nephritis in another. We believe that hepatitis may be produced by treponemes in early syphilitic infection. This syphilitic hepatitis can be differentiated from other forms of hepatitis, and we recommend dietary treatment and rest for these patients as well as specific penicillin treatment. Requests for reprints should be addressed to J.F., H-1430 Budapest, 3rd Department of Medicine, Semmelweis Medical University, Mezo Imre Ut 17,


Discussion The association of syphilis and liver disease may be fortuitous. In the past it may have been produced in some patients by arsphenamine therapy, which was very hepatotoxic. In other cases the patient may have been infected with hepatitis by injection during treatment. In other cases, a Herxheimer reaction following treatment may have led to liver damage. A viral aetiology was proposed by Papaevangeolu et al.,10 who pointed out that hepatitis-B virus may be sexually transmitted. We conclude, however, that in some cases of syphilis a characteristic hepatitis occurs at an early stage of the disease. Our suggestion is supported by the following findings in our cases of early syphilis: 1. An increase in serum-bilirubin concentration and hepatome-

galy are common clinical symptoms. 2. Routine laboratory data often indicate liver damage, including increased blood sedimentation, a positive colloid lability test, increase in S.G.O.T. and S.G.P.T. activity, raised bilirubin level, abnormal prothrombin-time and B.s.P. value, together with alterations in serum-protein levels. 3. The change in serum-immunoglobulins, especially in IgG and IgM concentration, resembles that found in virial hepatitis. 4. Our cases, with

exception, had definite histological evidence of hepatitis. The inflammation was partly of the focal necrotic type in the lobules of the liver and partly of proliferative character, in the walls of branches of central veins, arterioles, and portal veins. Inflammation of the central vein with fibrosis of its wall was especially common. These changes are characteristic, in our opinion, of early syphilitic liver damage. 5. We suggest that treponemes are involved in the pathogenesis of early syphilitic liver damage. In half of the cases with clinical evidence of liver damage, spirochaetes could be seen, not only in the necrotic foci but also in the walls of sinusoids and in the endothelial cells, as well as in the spaces between the liver cells. To our knowledge this is the first time that treponemes have been demonstrated by light and electron microscopy in syphilitic liver lesions at this

1. Hahn, R. D. Am. J. Syph. 1943, 27, 529. 2. Turner, T. B., Hardy, D. H., Newman, B. Br. J. vener. Dis. 1969, 45, 183. 3. Baker, A. I., Kaplan, M. M., Wolfe, H. J., McGowan, J. A. New Engl. J. Med. 1971, 284, 1422. 4. Lee, R. V., Thornton, G. F., Conn, H. J. ibid. p. 1423. 5. Parker, J. D. J. Br. J. vener. Dis. 1972, 48, 32. 6. Sarkany, I. Proc. R. Soc. Med. 1973, 66, 237. 7. Sehgal, V. M., Rege, V. L. Br. J. vener. Dis. 1974, 50, 237. 8. Mancini, G., Carbonara, A. D., Heremans, J. F. Immunochemistry, 1965,

2, 235. 9. 10.

Zugibe, F. I. Diagnostic Histochemistry; p. 318. St. Louis, Missouri, 1970. Papaevangeolu, G., Trichopoulos, D., Papoutsakis, G., Kremastinou, T., Pavlides, E. Br. J. vener. Dis. 1974, 50, 228.



Departments of Virology and Biological Chemistry, Laboratories, Beckenham, Kent, Department of Biochemistry, Charing Cross Hospital

Wellcome Research and

Medical School, London W6


stage of the disease. 6. The severity of immunological and glycoprotein abnormalities gradually decreased and then returned to normal after adequate antisyphilitic therapy with penicillin. Repeat


The surface hæmagglutinin and neuraminidase projections of influenza virus were removed from the viral envelope, purified, and relocated on the surface of unilamellar liposomes. The resulting structures were examined in the electron microscope and found to resemble the original virus. Units of both the viral hæmagglutinin and viral neuraminidase could be discerned. The name virosome is proposed for these new bodies.



THERE is increasing interest in the use of liposomes carriers of drugs’ and enzymes2 and in their potential

immunological adjutants. 34 Liposomess


as as


bodies consisting of aqueous dispersions of phospholipid in the form of either multi or unilamellar lipid bilayers.