- Email: [email protected]
Pathological and Epidemiological Aspects of Skin Lesions in Histoplasmosis
Zbl. Bakt. 285, 531-539 (1997) © Gustav Fischer Verlag, Jena
Pathological and Epidemiological Aspects of Skin Lesions in Histoplasmosis Observations in an AIDS Patient and Badgers Outside Endemic Areas of Histoplasmosis G. GROSSEl, F. STAIB*,]. RAPp2, H. RANG\ W. HEISE\ and L. KAUFMAW lInstitut fur Pathologie (Leiter: Prof. Dr. F. Niedobitek), Auguste-Viktoria-Krankenhaus, Berlin, Germany 2 Staatliches Tierarztliches Untersuchungsamt, Aulendorf, Germany 3 Tierhygienisches Institut, Freiburg i. Breisgau, Germany 4 II. Innere Klinik (Leiter: Prof. Dr. M. L'age), Auguste-Viktoria-Krankenhaus, Berlin, Ger many 5 Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, USA
Received August 12, 1996 . Accepted October 11, 1996
Summary As a consequence of HIV infection, histoplasmosis is increasingly occurring as an oppor tunistic infection with a systemic course outside histoplasmosis-endemic areas, e. g. in Eu rope. Accordingly, questions concerning the epidemiology of this mycosis arise. Two incidents involving histoplasmosis in man and badgers with prevailing involvement of the skin encouraged us to review the pathogenesis and epidemiology of this mycosis in Germany, where so far Histoplasma capsulatum has not been endemic. With a view to pre vention, attention is drawn to the avoidance of microfoci of H. capsulatum in the newly in troduced concept of biowaste and its composting plants in countries with modern waste management.
Introduction As a consequence of HIV infection, histoplasmosis as an opportunistic infection with a systemic course is increasingly occurring in histoplasmosis-endemic areas (16) as well as outside these areas, e. g. in Europe. Two incidents of histoplasmosis in man (10) and animals (19) with prevailing involvement of the skin encourage us to discuss * Former Chief, Mycology Unit, Robert Koch Institute, Berlin.
532
G. Grosse et al.
the pathogenesis and epidemiology of this mycosis in Germany, where so far Histo plasma capsulatum has not been endemic. Some of Rippon's comments on histoplas mosis , reflect the broad and deep experience of American experts with this mycosis: "Histoplasmosis is a very common granulomatous disease, caused by H. capsulatum; .. . histoplasmosis manifests itself in a bewildering array of clinical types and varia tions; ... it has been called the syphilis of the fungus world; ... with the exception of rare, accidental cutaneous inoculation, infection by H. capsula tum occurs by way of the lungs" (20). With a view to prevention, attention shall be drawn to the avoidance of microfoci of H. capsulatum (7) in the newly introduced concept of biowaste and its composting plants in countries with modern waste management (24, 25, 29).
Material and Methods A comparison will be made of skin lesions in an AIDS patient in Berlin and in wildlife badgers found in Germany. Both observations were published independently of one another (10, 19). For an understanding of the comparative studies, some substantial information on the two incidents are given. 1. A homosexual Indonesian, male, 55 y, living in Berlin for 20 years, became ill with histoplasmosis of the skin as the first opportunistic AIDS-defining infection; the CD4 lym-
Fig. 1. Skin lesions in the face of the AIDS patient caused by H. capsulatum. The thick crusts removed from the lesions, over w eeks were put into the biowaste container by the patient before the diagnosis of AIDS and histoplasmosis. Fig. 2. Ulcerated granuloma form ation in the skin of the head and neck of a badger caused by H. capsulatum.
Histoplasmosis of man and badger
533
phocyte count was 30/t-t1 (normal count 600-1250/t-t1). There were conspicuous desquamat ing, in part ulcerating, skin lesions over the face and shoulder region; the lungs and other organs were free of symptoms. Budding yeast-like cells found microscopically in native preparations as well as in histological sections (HE, PAS, and Grocott stains) were con firmed as H. capsulatum var. capsulatum by the fluorescent antibody technique (FAT) (15) as well as by culture (10) (Figs. 1, 3a, b, 5). 2. At the Governmental Veterinarian Office in Aulendorf/Oberschwaben and the Insti tute of Veterinary Hygiene in FreiburglBreisgau, i. e. in the southern parts of Germany, al most contemporarily conspicuous granulomatous skin lesions were found in 3 cases in wild badgers. No other organs were involved. The animals were adults, one male and two females and were victims of car accidents; their corpses were confiscated in view of an examination for rabies and because of the conspicuous skin lesions. Histological studies revealed yeast like fungi whose morphology was characteristic of H. capsulatum var. capsulatum (cultural examinations for fungi were not performed) (19). For confirmation, comparative examina tions of tissue sections (HE, PAS, and Grocott stains) (Figs. 4a,b) were carried out together with those from AIDS patients suffering from histoplasmosis (Figs. 3a,b) at the Institute of Pathology of the Auguste-Viktoria-Hospital in Berlin. Those were repeatedly diagnosed by Grosse and culturally confirmed by Staib (10, 28) as H. capsulatum. In addition, unstained tissue sections from skin lesions of the badger were sent for FAT to Kaufman at the CDC, AtlantalUSA (15), where the yeasts were identified as H. capsulatum var. capsulatum. Results Comparative microscopic examination of the histological sections and native pre parations from skin lesions (and crusts) of the AIDS patient (10) and those of the bad gers (19) revealed the following: 1. In the skin ulcers of face (Fig. 1) and shoulder of the AIDS patient, the histologi cal findings revealed a chronic inflammation with non-specific granulation tissue in the base of the ulcer. In the cytoplasm of numerous macrophages of the granulation tissue, many yeast-like round-oval cells (3-4 t-tm) were found. These cells appeared stained by PAS because of the unstained cell wall much smaller than after Grocott staining (Fig. 3b). Also in the crusts of fibrin found as the uppermost layer on the skin lesions in the face, many macrophages with intracellular fungal cells beside freely occurring cells could be recognized. Also in the uppermost layers of the crusts, apart from the yeast-like cells, neither mycelium formation nor transient forms between yeast and my celial form of H. capsulatum could be recognized (Fig. 5). 2. In tumour-like exulcerated skin infiltrates of the badgers (Fig. 2), the histological findings presented also a chronic inflammation with a non-specific granulation tissue with numerous, partly crowded, macrophages containing in their cytoplasm many round-oval yeast-like cells appearing larger in size if stained by Grocott stain instead of PAS (Figs. 4a,b). These cells corresponded to the yeast form of H. capsulatum var. capsulatum having a size of 3-4 (..lm. 3. Comparison of the microscopic tissue sections of man and animals revealed, for the cells of the pathogenic agent, a uniform picture of round-oval yeast-like cells cor responding to the tissue form of H. capsulatum var. capsula tum (Figs. 3, 4). Histologi cally, in connection with the ulcerating defects of tissue, there were similar changes combined with an identical morphology of the pathogen and characteristic intracellu lar manifestation within the numerous macrophages of the chronic granulation tissue as an inflammatory reaction of non-specific character. There was a difference in the extension of the skin lesions; probably, in the badgers, the lesions could spread over a
534
G. Grosse et al.
long time without any treatment. The intracellular distribution of the pathogen found in non-specific granulation tissue of ulcerating skin defects of the AIDS patient and the badgers presented a uniform picture. Undoubtedly, an immunodeficiency has to be as sumed to exist also in the badgers.
Histoplasmosis of man and badger
535
Discussion H. capsulatum in skin lesions of man and animal Our pathogenetic and epidemiologic interest shall be devoted to the conspicuous in volvement of the skin by H. capsulatum var. capsulatum as first AIDS-defining oppor tunistic infection in a HIV-positive man with a decreased CD4 lymphocyte count of 30/!1l and to a similar conspicuous involvement of the skin in 3 wild badgers with an unknown immune status (10, 19). In the USA, in 26 out of 239 HIV-infected persons suffering from disseminated histoplasmosis (i. e. 11 %) skin lesions by H. capsulatum were detected by Cohen et al. (5). Only in a few of these patients an inconspicuous status of the lungs was found (5), like in the two incidents of the present report, i. e. the AIDS patient and the 3 badgers (as far as it could be detected post mortem) (10, 19). Cohen found the skin lesions of histoplasmosis in AIDS patients to be most dif ferent; these partly exulcerated skin lesions were preferably found on the arms, in the face, and on the trunk (5), like in the AIDS patient and in the badgers described in this report (Figs. 1, 2). In the crusts of the skin lesions of the AIDS patient as well as in the uppermost layers of the skin lesions of the 3 badgers H. capsulatum was found in large quantities in the so-called yeast or tissue form (Figs. 3-5). A presupposition for this morphological phase of H. capsulatum are a temperature of 37°C unless, there are spe cial nutrient conditions, i. e. the sulfhydryl (SH) groups, e. g. cysteine, then the temper (4, 11-13, 17,22). Because during the growth of H. capsu ature may be below latum and its persistence in the yeast form in the uppermost layers of the crusts on the skin lesions (of the AIDS patient) with great probability the temperature was below 37°C, the following questions arise: Is in each tissue with normal blood supply in man and animal, the concentration of SH groups physiologically sufficient to secure its re quirements, e. g. for the formation of the yeast forms, or must the striking involvement of the skin by H. capsulatum in the yeast form be interpreted as a result of inflamma tion and repair under conditions of a disturbed T-cell mediated immunity (21). Fur ther studies in this field are needed.
3rc
Fig. 3a, b. Tissue section out of the skin ulcer from the face of the AIDS patient. Histologi cally, beside squamous epithelial formation of the corneum, cell detritus and fibrin, several cells mainly macrophages loaded by masses of fungi can be detected (arrow). In the cyto plasm of the macrophages, groups of round-oval budding yeast-like cells corresponding to the yeast form of H. capsula tum var. capsulatum can be recognized due to the silver stain ing (Fig. 3a: Grocott; objective X 40) and as smaller ones by the unstained cell wall appear ing like a capsule (Fig. 3b: PAS; objective x 40). Fig. 4a, b. Tissue section of the ulcerated skin lesions of histoplasmosis in the badger. In the non-specific granulation tissue, numerous macrophages contain in their cytoplasm many round-oval yeast-like cells (arrow) appearing in normal size after Grocott (Fig.4a; objec tive X 40) and PAS (Fig. 4b; objective X 40) staining as small particles with an unstaind cell wall, corresponding to H. capsula tum var. capsula tum. Fig. 5. Crusts of the skin lesions in the face of the AIDS patient were suspended in 0.85% NaClsolution. Among detritus of skin tissue, round-oval cells (ca. 3-4 Itm), partly in a bud ding stage; corresponding in shape and size to the so-called yeast or tissue form of H. cap sulatum var. capsulatum. Neither mycelium formation nor transient forms between yeast and mycelial form of H. capsulatum can be recognized. (Native preparation of crushed crusts; objective X 40) (see also Figs. 3,4).
536
G. Grosse et al.
Histoplasmosis of the badger In Aiello's review of the comparative ecology of respiratory mycotic disease agents in 1967 (1) he reported that a large variety of both feral and domesticated animals had been victims of histoplasmosis. Among those studied was only one case in a badger in Swit zerland in 1961 (3). In this overview, next to man, the dog was found to be the animal most susceptible to infection by H. capsulatum (1). Since the first reported histoplasmo sis case in a badger in Switzerland in 1961, additional cases were reported in 1992 from Germany (19) and Denmark (14). Despite their life close to other animal species (e. g. the fox) as sole wild animals, an extraordinary predisposition of this animal must be sup posed for this systemic mycosis. According to the observations of Rapp et aI., the bad ger lives in secrecy and is a robust animal species. Autopsy evidence suggests that it on ly rarely harbors specific diseases (19). Various circumstances aggravate the search for H. capsulatum in this animal's environment. Because of the lack of differential culture media, the cultural isolation of H. capsulatum from soil, plant material, etc. is still time consuming (7,16,20,28). Because the badger is an omnivore, it will be difficult to fol low the course of infection and establish its source e. g. a specific feeding habit. It must be supposed that in badgers suffering from histoplasmosis immunodeficien cies of various origin may be involved pathogenetically. How far a constant intake of poisoned nourishment (e. g. pesticides) by the badger as shown in Great Britain (6) led to chronic damage, e. g. disturbances of kidney function with subsequent immunodefi ciencies was not reported. Jensen et al. who reported on the histoplasmosis case of a badger with skin lesions from Denmark (14) suggested that the heavy infection of the badger by the hookworm Uncinaria spp. may have contributed to its state of emacia tion, secondarily lowering its resistance to the H. capsulatum infection. In the almost simultaneous report on the 3 badgers from Germany, no observations that would ex plain an immunodeficiency were mentioned by Rapp et al. (19). We hypothesize that for the pathogenesis of histoplasmosis of the badger, similar conditions exist as in the case of cryptococcosis of HIV-infected persons consisting of the pathogen's habitat (2, 23), and an immunodeficiency of the exposed host, charac terized by a heavily decreased CD4lymphocyte count (2). In the past, there were cases of systemic C. neoformans infections in wild animals (e. g. fox, mouse, etc.) in which due to external circumstances, the responsible immu nodeficiency could not be detected (27, 30). In this connection, earlier observations made by the authors (26) in animal experiments (white mouse, NMRI) had drawn at tention to the possibility of acute or chronic poisoning leading to uremic stages com bined with immunodeficiency (8, 9). It could be shown that poisoning leading to a reparable two-day disturbance of the kidney function with uremia, a latent asympto matic C. neoformans infection (by a C. neoformans-strain of very low virulence) could be transferred to a fast and a heavy exacerbation of the infection with a massive in volvement of the brain (9, 26). Therefore, in future studies on the origin of predispos ing immunodeficiencies in such animals, attention should also be drawn to chronic poi soning, preferably via nourishment (6,14, 19). Avoidance of microfoci of H. capsulatum in biowaste and compost By the examples of the AIDS patient from Berlin and the badgers from Germany and Denmark, attention is drawn to the fact that also outside the endemic areas of histo plasmosis, a spreading of H. capsulatum by particles of crusts of the skin lesions in the direct environment of man and animal is possible (10, 19). With the observations of
Histoplasmosis of man and badger
537
Di Salvo, USA attention shall also be drawn to so-called microfoci of H. capsulatum, i. e. locally limited endemic areas, which may occur everywhere (7). Such microfoci could also be assumed in the habitat of a badger, e. g. in the soil of a forest, in badger burrows, etc. Because the AIDS patient to whom this report refers had mentioned that before the diagnosis of histoplasmosis in his case was made, over weeks, he daily re moved the crusts of the skin lesions from his face and put them into the biological waste container together with paper handkerchiefs, it must be assumed that in this way, a contamination or colonization of biowaste by H. capsulatum could have taken place (10, 24, 25, 29). Because in Germany, no habitats of H. capsulatum like in drop pings of bats and birds as in the USA have been found so far, the possibility of an im portation of this fungus to Europe by AIDS patients from overseas endemic areas can not be excluded (10, 28). The idea of possible microfoci of H. capsulatum in collected biological wastes and areas of composting plants does not seem to be irrelevant if taking into account the great variability of biowaste (protein-rich food, carbohydrates, lipids, etc.) (24, 25, 29). For this reason, brain-heart-infusion agar (BHI), a nutrient agar used world-wide to cultivate agents of systemic mycoses, among which H. capsulatum should be men tioned (16). A micro focus of H. capsulatum in the area of a compo sting plant for biowaste could become of great epidemiological significance, especially for wildlife animals which in their search for feed (18), in addition to the possibility of contamination by opportu nistic pathogens, could also be exposed to poisoning (e. g. by preservatives, pesticides, etc.). Since the badger as an omnivore was frequently seen in the environment of col lected biowastes and in the environment of composting plants (unpublished data), the foregoing observations should be understood as a new epidemiological situation, also in the field of the epidemiology of systemic mycoses of man and animal outside the classic endemic areas of histoplasmosis. To supervise the new field "Health risks from decay of organic wastes and compost; proposals for mycological control", Staib has made twelve proposals for mycological control among which in this context the two proposals, Nos. XI and XII, are of inter est, i. e. " ... surveillance should include wildlife animals (e. g. the fox, the badger, the crow, etc.) and pests (e. g. the cockroach) in the immediate environment of compost ing plants" and " ... discussion is needed about the extent to which materials should be excluded from biological waste container ... materials from AIDS patients conta minated with C. neoformans or H. capsulatum, etc." (25,29). In order to solve such increasing future tasks in the surveillance of wildlife animals, the corresponding activities of the Institute for Zoo biology and Wildlife Research in Berlin-Friedrichsfelde are more than justified (18).
Conclusion
It was found that in the epidemiology of histoplasmosis in connection with the HIV infection of man attention must also be drawn to microfoci of H. capsulatum in bio logical waste and wildlife animals as demonstrated by histoplasmosis of the badger outside endemic areas as shown in Germany. In view of a preventive health protection and basic research in the field of systemic mycoses, success will only be possible by in tense cooperation of veterinary and human medicine as well as the various fields of bi ology including waste and soil microbiology.
538
G. Grosse et al.
References 1. Aiello, L.: Comparative ecology of respiratory mycotic disease agents. Bact. Rev. 31 (1967) 6-24 2. Arasteh, K., F. Staib, G. Grosse, U. Futh, and M. L'age: Cryptococcosis in HIV infec tion of man: an epidemiological and immunological indicator? Zbl. Bakt. 284 (1996) 153-163 3. Burgisser, H. , R. Frankenhauser, W. Kaplan, K. Klingler und H.]. Scholer: Mykose bei einem Dachs in der Schweiz: Histologisch Histoplasmose. Parhol. Microbiol. 24 (1961) 794-802 4. Campbell, C. c.: Reverting Histoplasma capsulatum to the yeast phase. J. Bact. 54 (1947) 263-264 5. Cohen, P. R., D. E. Bank, D. N. Silvers, and M. E. Grossman: Cutaneous lesions of dis seminated histoplasmosis in human immunodeficiency virus-infected patients. J. Amer. Acad. Dermatol. 23 (1990) 422-428 6. Cresswell, P., S. Harris, and D.]. Jefferies: The history, distribution, status and habitat requirements of the badger in Britain. pp. 8, 10. Publicity Services Branch, Nature Con servancy Council, Northminster House, Peterborough PEllUA (1990) 7. Di Salvo, A. F. and W. M. Johnson: Histoplasmosis in South Carolina: support for the microfocus concept. Am. J. Epidemiol. 109 (1979) 480-492 8. Fromtling, R. A., Anne, M. Fromtling, F. Staib, and Sybille Muller: Effect of uremia on lymphocyte transformation and chemiluminescence by spleen cells of normal and Cryp tococcus neoformans-infected mice. Infect. and Immun. 32 (1981) 1073-1078 9. Grosse, G., S. K. Mishra, and F. Staib: Selective involvement of the brain in experimen tal murine cryptococcosis. II. Histopathological observations. Zbl. Bakt. Hyg. 1. Abt. Orig. A 233 (1975) 106-122 10. Grosse, G., W. Heise und F. Staib: Hisroplasmose der Haut als opportunistische Erstin fektion bei AIDS. Dtsch. Med. Wschr. 118 (1993) 1555-1560 11. Howard, D. H.: Intracellular growth of Histoplasma capsulatum. J. Bacteriol. 89 (1965) 518-523 12. Howard, D. H.: Effect of temperature of intracellular growth of Histoplasma capsula tum. J. Bacterial. 93 (1967) 438-444 13. Howard, D. H., N. Dabrowa, V. Otto, and]. Rhodes: Cysteine transport and sulfite re ductase activity in a germinationdefective mutant of Histoplasma capsulatum. J. Bacte riol. 141 (1980) 417-421 14.Jensen, H. E., B. Bloch, P. Henriksen, H. H. Dietz, H. Schonheyder, and L. Kaufman: Disseminated histoplasmosis in a badger (Meles meles) in Denmark. APMIS 100 (1992) 586-592 15. Kaufman, L.: Immunohistologic diagnosis of systemic mycoses: an update. Eur. J. Epi demiol. 8 (1992) 377-382 16.Kwon-Chung, K.]. and J.E. Bennett: Medical Mycology. Lea & Febiger, Philadel phia-London (1992) 17. Medoff, G., G. S. Kobayashi, A. Painter, and S. Travis: Morphogenesis and pathogeni city of Histoplasma capsulatum. Infect. Immun. 55 (1987) 1355-1358 18. Priemer, J. and E. Lux: Helminths of badgers in Germany. Parassitologia (Roma) (Ab stracts) 38 (1996) 20 19. Rapp, J., A. Lofqvist, E. Breuer und H. Rang: SproGpilze als Ursache von Hautgranulo men beim Dachs in Siiddeutschland. Tierarztl. Umschau 47 (1992) 451-454 20. Rippon,]. w.: Medical Mycology. 3rd Ed., p. 405. W. B. Saunders Comp., Philadelphia (1988) 21. Robbins, St. L. and R. S. Cotran: Pathologic basis of disease. 2nd Ed., pp. 69-76. W. B. Saunders Comp., Philadelphia, London, Toronto (1979) 22. Scherr, G. H.: Studies on the dimorphism of Histoplasma capsulatum. 1. The roles of SH groups and incubation temperature. Exp. Cell Res. 12 (1957) 92-107
Histoplasmosis of man and badger
539
23 . Staib, F.: Cryptococcus neoformans and Cryptococcosis. Peculiarities and challenge: Opening lecture. J. Mycol. Med. 4 (1994) 56-60 24. Staib, F.: Bioabfall aus medizinisch-mykologischer Sicht. Bundesgesundheitsbl. 35 (1992) 21-26 25. Staib, F.: Editorial: Fungi in biowaste and compost. Medical-mycological aspects and proposals. Zbl. Bakt. 285 (1996) 1-4 26. Staib, F., S. K. Mishra, G. Grosse, and Th. Abel: Ocular cryptococcosis - experimental and clinical observations. Zbl. Bakt. Hyg. I. Abt. Orig. A 237 (1977) 378-394 27. Staib, F., W. Weller, S. Brem, R. Schindlmayr, and E. Schmittdiel: A Cryptococcus neo formans strain from the brain of a wildlife fox (Vulpes vulpes) suspected of rabies: My cological observations and comments. Zbl. Bakt. Hyg. A 260 (1985) 566-571 28. Staib, F. and G. Grosse: Brown-red pigment formation by the mycelial phase of a clini cal isolate of Histoplasma capsulatum on Staib agar. A preliminary report. Zbl. Bakt. 283 (1996)515-521 29. Summerbell, R. c., F. Staib, D. G. Ahearn et al.: Household hyphomycetes and other in door fungi. J. Med. Vet. Mycol. 32 (Suppl. 1) (1994) 277-286 30. Weller, w., S. Brem, R. Schindlmayr und E. Schmittdiel: Cryptococcose bei einem Rot fuchs (Vulpes vulpes). Berl. Munch. tierarztl. Wschr. 98 (1985) 14-15
Prof. Dr. med. G. Grosse, Institut fur Pathologie, Auguste-Viktoria-Krankenhaus, Ru bensstraRe 125, D-12157 Berlin, Germany Prof. Dr. med. Dr. F. Staib, BrentanostraRe 26, D-12163 Berlin, Germany
35
Zbl. Bakt 285/4
Pathological and Epidemiological Aspects of Skin Lesions in Histoplasmosis Observations in an AIDS Patient and Badgers Outside Endemic Areas of Histoplasmosis G. GROSSEl, F. STAIB*,]. RAPp2, H. RANG\ W. HEISE\ and L. KAUFMAW lInstitut fur Pathologie (Leiter: Prof. Dr. F. Niedobitek), Auguste-Viktoria-Krankenhaus, Berlin, Germany 2 Staatliches Tierarztliches Untersuchungsamt, Aulendorf, Germany 3 Tierhygienisches Institut, Freiburg i. Breisgau, Germany 4 II. Innere Klinik (Leiter: Prof. Dr. M. L'age), Auguste-Viktoria-Krankenhaus, Berlin, Ger many 5 Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, USA
Received August 12, 1996 . Accepted October 11, 1996
Summary As a consequence of HIV infection, histoplasmosis is increasingly occurring as an oppor tunistic infection with a systemic course outside histoplasmosis-endemic areas, e. g. in Eu rope. Accordingly, questions concerning the epidemiology of this mycosis arise. Two incidents involving histoplasmosis in man and badgers with prevailing involvement of the skin encouraged us to review the pathogenesis and epidemiology of this mycosis in Germany, where so far Histoplasma capsulatum has not been endemic. With a view to pre vention, attention is drawn to the avoidance of microfoci of H. capsulatum in the newly in troduced concept of biowaste and its composting plants in countries with modern waste management.
Introduction As a consequence of HIV infection, histoplasmosis as an opportunistic infection with a systemic course is increasingly occurring in histoplasmosis-endemic areas (16) as well as outside these areas, e. g. in Europe. Two incidents of histoplasmosis in man (10) and animals (19) with prevailing involvement of the skin encourage us to discuss * Former Chief, Mycology Unit, Robert Koch Institute, Berlin.
532
G. Grosse et al.
the pathogenesis and epidemiology of this mycosis in Germany, where so far Histo plasma capsulatum has not been endemic. Some of Rippon's comments on histoplas mosis , reflect the broad and deep experience of American experts with this mycosis: "Histoplasmosis is a very common granulomatous disease, caused by H. capsulatum; .. . histoplasmosis manifests itself in a bewildering array of clinical types and varia tions; ... it has been called the syphilis of the fungus world; ... with the exception of rare, accidental cutaneous inoculation, infection by H. capsula tum occurs by way of the lungs" (20). With a view to prevention, attention shall be drawn to the avoidance of microfoci of H. capsulatum (7) in the newly introduced concept of biowaste and its composting plants in countries with modern waste management (24, 25, 29).
Material and Methods A comparison will be made of skin lesions in an AIDS patient in Berlin and in wildlife badgers found in Germany. Both observations were published independently of one another (10, 19). For an understanding of the comparative studies, some substantial information on the two incidents are given. 1. A homosexual Indonesian, male, 55 y, living in Berlin for 20 years, became ill with histoplasmosis of the skin as the first opportunistic AIDS-defining infection; the CD4 lym-
Fig. 1. Skin lesions in the face of the AIDS patient caused by H. capsulatum. The thick crusts removed from the lesions, over w eeks were put into the biowaste container by the patient before the diagnosis of AIDS and histoplasmosis. Fig. 2. Ulcerated granuloma form ation in the skin of the head and neck of a badger caused by H. capsulatum.
Histoplasmosis of man and badger
533
phocyte count was 30/t-t1 (normal count 600-1250/t-t1). There were conspicuous desquamat ing, in part ulcerating, skin lesions over the face and shoulder region; the lungs and other organs were free of symptoms. Budding yeast-like cells found microscopically in native preparations as well as in histological sections (HE, PAS, and Grocott stains) were con firmed as H. capsulatum var. capsulatum by the fluorescent antibody technique (FAT) (15) as well as by culture (10) (Figs. 1, 3a, b, 5). 2. At the Governmental Veterinarian Office in Aulendorf/Oberschwaben and the Insti tute of Veterinary Hygiene in FreiburglBreisgau, i. e. in the southern parts of Germany, al most contemporarily conspicuous granulomatous skin lesions were found in 3 cases in wild badgers. No other organs were involved. The animals were adults, one male and two females and were victims of car accidents; their corpses were confiscated in view of an examination for rabies and because of the conspicuous skin lesions. Histological studies revealed yeast like fungi whose morphology was characteristic of H. capsulatum var. capsulatum (cultural examinations for fungi were not performed) (19). For confirmation, comparative examina tions of tissue sections (HE, PAS, and Grocott stains) (Figs. 4a,b) were carried out together with those from AIDS patients suffering from histoplasmosis (Figs. 3a,b) at the Institute of Pathology of the Auguste-Viktoria-Hospital in Berlin. Those were repeatedly diagnosed by Grosse and culturally confirmed by Staib (10, 28) as H. capsulatum. In addition, unstained tissue sections from skin lesions of the badger were sent for FAT to Kaufman at the CDC, AtlantalUSA (15), where the yeasts were identified as H. capsulatum var. capsulatum. Results Comparative microscopic examination of the histological sections and native pre parations from skin lesions (and crusts) of the AIDS patient (10) and those of the bad gers (19) revealed the following: 1. In the skin ulcers of face (Fig. 1) and shoulder of the AIDS patient, the histologi cal findings revealed a chronic inflammation with non-specific granulation tissue in the base of the ulcer. In the cytoplasm of numerous macrophages of the granulation tissue, many yeast-like round-oval cells (3-4 t-tm) were found. These cells appeared stained by PAS because of the unstained cell wall much smaller than after Grocott staining (Fig. 3b). Also in the crusts of fibrin found as the uppermost layer on the skin lesions in the face, many macrophages with intracellular fungal cells beside freely occurring cells could be recognized. Also in the uppermost layers of the crusts, apart from the yeast-like cells, neither mycelium formation nor transient forms between yeast and my celial form of H. capsulatum could be recognized (Fig. 5). 2. In tumour-like exulcerated skin infiltrates of the badgers (Fig. 2), the histological findings presented also a chronic inflammation with a non-specific granulation tissue with numerous, partly crowded, macrophages containing in their cytoplasm many round-oval yeast-like cells appearing larger in size if stained by Grocott stain instead of PAS (Figs. 4a,b). These cells corresponded to the yeast form of H. capsulatum var. capsulatum having a size of 3-4 (..lm. 3. Comparison of the microscopic tissue sections of man and animals revealed, for the cells of the pathogenic agent, a uniform picture of round-oval yeast-like cells cor responding to the tissue form of H. capsulatum var. capsula tum (Figs. 3, 4). Histologi cally, in connection with the ulcerating defects of tissue, there were similar changes combined with an identical morphology of the pathogen and characteristic intracellu lar manifestation within the numerous macrophages of the chronic granulation tissue as an inflammatory reaction of non-specific character. There was a difference in the extension of the skin lesions; probably, in the badgers, the lesions could spread over a
534
G. Grosse et al.
long time without any treatment. The intracellular distribution of the pathogen found in non-specific granulation tissue of ulcerating skin defects of the AIDS patient and the badgers presented a uniform picture. Undoubtedly, an immunodeficiency has to be as sumed to exist also in the badgers.
Histoplasmosis of man and badger
535
Discussion H. capsulatum in skin lesions of man and animal Our pathogenetic and epidemiologic interest shall be devoted to the conspicuous in volvement of the skin by H. capsulatum var. capsulatum as first AIDS-defining oppor tunistic infection in a HIV-positive man with a decreased CD4 lymphocyte count of 30/!1l and to a similar conspicuous involvement of the skin in 3 wild badgers with an unknown immune status (10, 19). In the USA, in 26 out of 239 HIV-infected persons suffering from disseminated histoplasmosis (i. e. 11 %) skin lesions by H. capsulatum were detected by Cohen et al. (5). Only in a few of these patients an inconspicuous status of the lungs was found (5), like in the two incidents of the present report, i. e. the AIDS patient and the 3 badgers (as far as it could be detected post mortem) (10, 19). Cohen found the skin lesions of histoplasmosis in AIDS patients to be most dif ferent; these partly exulcerated skin lesions were preferably found on the arms, in the face, and on the trunk (5), like in the AIDS patient and in the badgers described in this report (Figs. 1, 2). In the crusts of the skin lesions of the AIDS patient as well as in the uppermost layers of the skin lesions of the 3 badgers H. capsulatum was found in large quantities in the so-called yeast or tissue form (Figs. 3-5). A presupposition for this morphological phase of H. capsulatum are a temperature of 37°C unless, there are spe cial nutrient conditions, i. e. the sulfhydryl (SH) groups, e. g. cysteine, then the temper (4, 11-13, 17,22). Because during the growth of H. capsu ature may be below latum and its persistence in the yeast form in the uppermost layers of the crusts on the skin lesions (of the AIDS patient) with great probability the temperature was below 37°C, the following questions arise: Is in each tissue with normal blood supply in man and animal, the concentration of SH groups physiologically sufficient to secure its re quirements, e. g. for the formation of the yeast forms, or must the striking involvement of the skin by H. capsulatum in the yeast form be interpreted as a result of inflamma tion and repair under conditions of a disturbed T-cell mediated immunity (21). Fur ther studies in this field are needed.
3rc
Fig. 3a, b. Tissue section out of the skin ulcer from the face of the AIDS patient. Histologi cally, beside squamous epithelial formation of the corneum, cell detritus and fibrin, several cells mainly macrophages loaded by masses of fungi can be detected (arrow). In the cyto plasm of the macrophages, groups of round-oval budding yeast-like cells corresponding to the yeast form of H. capsula tum var. capsulatum can be recognized due to the silver stain ing (Fig. 3a: Grocott; objective X 40) and as smaller ones by the unstained cell wall appear ing like a capsule (Fig. 3b: PAS; objective x 40). Fig. 4a, b. Tissue section of the ulcerated skin lesions of histoplasmosis in the badger. In the non-specific granulation tissue, numerous macrophages contain in their cytoplasm many round-oval yeast-like cells (arrow) appearing in normal size after Grocott (Fig.4a; objec tive X 40) and PAS (Fig. 4b; objective X 40) staining as small particles with an unstaind cell wall, corresponding to H. capsula tum var. capsula tum. Fig. 5. Crusts of the skin lesions in the face of the AIDS patient were suspended in 0.85% NaClsolution. Among detritus of skin tissue, round-oval cells (ca. 3-4 Itm), partly in a bud ding stage; corresponding in shape and size to the so-called yeast or tissue form of H. cap sulatum var. capsulatum. Neither mycelium formation nor transient forms between yeast and mycelial form of H. capsulatum can be recognized. (Native preparation of crushed crusts; objective X 40) (see also Figs. 3,4).
536
G. Grosse et al.
Histoplasmosis of the badger In Aiello's review of the comparative ecology of respiratory mycotic disease agents in 1967 (1) he reported that a large variety of both feral and domesticated animals had been victims of histoplasmosis. Among those studied was only one case in a badger in Swit zerland in 1961 (3). In this overview, next to man, the dog was found to be the animal most susceptible to infection by H. capsulatum (1). Since the first reported histoplasmo sis case in a badger in Switzerland in 1961, additional cases were reported in 1992 from Germany (19) and Denmark (14). Despite their life close to other animal species (e. g. the fox) as sole wild animals, an extraordinary predisposition of this animal must be sup posed for this systemic mycosis. According to the observations of Rapp et aI., the bad ger lives in secrecy and is a robust animal species. Autopsy evidence suggests that it on ly rarely harbors specific diseases (19). Various circumstances aggravate the search for H. capsulatum in this animal's environment. Because of the lack of differential culture media, the cultural isolation of H. capsulatum from soil, plant material, etc. is still time consuming (7,16,20,28). Because the badger is an omnivore, it will be difficult to fol low the course of infection and establish its source e. g. a specific feeding habit. It must be supposed that in badgers suffering from histoplasmosis immunodeficien cies of various origin may be involved pathogenetically. How far a constant intake of poisoned nourishment (e. g. pesticides) by the badger as shown in Great Britain (6) led to chronic damage, e. g. disturbances of kidney function with subsequent immunodefi ciencies was not reported. Jensen et al. who reported on the histoplasmosis case of a badger with skin lesions from Denmark (14) suggested that the heavy infection of the badger by the hookworm Uncinaria spp. may have contributed to its state of emacia tion, secondarily lowering its resistance to the H. capsulatum infection. In the almost simultaneous report on the 3 badgers from Germany, no observations that would ex plain an immunodeficiency were mentioned by Rapp et al. (19). We hypothesize that for the pathogenesis of histoplasmosis of the badger, similar conditions exist as in the case of cryptococcosis of HIV-infected persons consisting of the pathogen's habitat (2, 23), and an immunodeficiency of the exposed host, charac terized by a heavily decreased CD4lymphocyte count (2). In the past, there were cases of systemic C. neoformans infections in wild animals (e. g. fox, mouse, etc.) in which due to external circumstances, the responsible immu nodeficiency could not be detected (27, 30). In this connection, earlier observations made by the authors (26) in animal experiments (white mouse, NMRI) had drawn at tention to the possibility of acute or chronic poisoning leading to uremic stages com bined with immunodeficiency (8, 9). It could be shown that poisoning leading to a reparable two-day disturbance of the kidney function with uremia, a latent asympto matic C. neoformans infection (by a C. neoformans-strain of very low virulence) could be transferred to a fast and a heavy exacerbation of the infection with a massive in volvement of the brain (9, 26). Therefore, in future studies on the origin of predispos ing immunodeficiencies in such animals, attention should also be drawn to chronic poi soning, preferably via nourishment (6,14, 19). Avoidance of microfoci of H. capsulatum in biowaste and compost By the examples of the AIDS patient from Berlin and the badgers from Germany and Denmark, attention is drawn to the fact that also outside the endemic areas of histo plasmosis, a spreading of H. capsulatum by particles of crusts of the skin lesions in the direct environment of man and animal is possible (10, 19). With the observations of
Histoplasmosis of man and badger
537
Di Salvo, USA attention shall also be drawn to so-called microfoci of H. capsulatum, i. e. locally limited endemic areas, which may occur everywhere (7). Such microfoci could also be assumed in the habitat of a badger, e. g. in the soil of a forest, in badger burrows, etc. Because the AIDS patient to whom this report refers had mentioned that before the diagnosis of histoplasmosis in his case was made, over weeks, he daily re moved the crusts of the skin lesions from his face and put them into the biological waste container together with paper handkerchiefs, it must be assumed that in this way, a contamination or colonization of biowaste by H. capsulatum could have taken place (10, 24, 25, 29). Because in Germany, no habitats of H. capsulatum like in drop pings of bats and birds as in the USA have been found so far, the possibility of an im portation of this fungus to Europe by AIDS patients from overseas endemic areas can not be excluded (10, 28). The idea of possible microfoci of H. capsulatum in collected biological wastes and areas of composting plants does not seem to be irrelevant if taking into account the great variability of biowaste (protein-rich food, carbohydrates, lipids, etc.) (24, 25, 29). For this reason, brain-heart-infusion agar (BHI), a nutrient agar used world-wide to cultivate agents of systemic mycoses, among which H. capsulatum should be men tioned (16). A micro focus of H. capsulatum in the area of a compo sting plant for biowaste could become of great epidemiological significance, especially for wildlife animals which in their search for feed (18), in addition to the possibility of contamination by opportu nistic pathogens, could also be exposed to poisoning (e. g. by preservatives, pesticides, etc.). Since the badger as an omnivore was frequently seen in the environment of col lected biowastes and in the environment of composting plants (unpublished data), the foregoing observations should be understood as a new epidemiological situation, also in the field of the epidemiology of systemic mycoses of man and animal outside the classic endemic areas of histoplasmosis. To supervise the new field "Health risks from decay of organic wastes and compost; proposals for mycological control", Staib has made twelve proposals for mycological control among which in this context the two proposals, Nos. XI and XII, are of inter est, i. e. " ... surveillance should include wildlife animals (e. g. the fox, the badger, the crow, etc.) and pests (e. g. the cockroach) in the immediate environment of compost ing plants" and " ... discussion is needed about the extent to which materials should be excluded from biological waste container ... materials from AIDS patients conta minated with C. neoformans or H. capsulatum, etc." (25,29). In order to solve such increasing future tasks in the surveillance of wildlife animals, the corresponding activities of the Institute for Zoo biology and Wildlife Research in Berlin-Friedrichsfelde are more than justified (18).
Conclusion
It was found that in the epidemiology of histoplasmosis in connection with the HIV infection of man attention must also be drawn to microfoci of H. capsulatum in bio logical waste and wildlife animals as demonstrated by histoplasmosis of the badger outside endemic areas as shown in Germany. In view of a preventive health protection and basic research in the field of systemic mycoses, success will only be possible by in tense cooperation of veterinary and human medicine as well as the various fields of bi ology including waste and soil microbiology.
538
G. Grosse et al.
References 1. Aiello, L.: Comparative ecology of respiratory mycotic disease agents. Bact. Rev. 31 (1967) 6-24 2. Arasteh, K., F. Staib, G. Grosse, U. Futh, and M. L'age: Cryptococcosis in HIV infec tion of man: an epidemiological and immunological indicator? Zbl. Bakt. 284 (1996) 153-163 3. Burgisser, H. , R. Frankenhauser, W. Kaplan, K. Klingler und H.]. Scholer: Mykose bei einem Dachs in der Schweiz: Histologisch Histoplasmose. Parhol. Microbiol. 24 (1961) 794-802 4. Campbell, C. c.: Reverting Histoplasma capsulatum to the yeast phase. J. Bact. 54 (1947) 263-264 5. Cohen, P. R., D. E. Bank, D. N. Silvers, and M. E. Grossman: Cutaneous lesions of dis seminated histoplasmosis in human immunodeficiency virus-infected patients. J. Amer. Acad. Dermatol. 23 (1990) 422-428 6. Cresswell, P., S. Harris, and D.]. Jefferies: The history, distribution, status and habitat requirements of the badger in Britain. pp. 8, 10. Publicity Services Branch, Nature Con servancy Council, Northminster House, Peterborough PEllUA (1990) 7. Di Salvo, A. F. and W. M. Johnson: Histoplasmosis in South Carolina: support for the microfocus concept. Am. J. Epidemiol. 109 (1979) 480-492 8. Fromtling, R. A., Anne, M. Fromtling, F. Staib, and Sybille Muller: Effect of uremia on lymphocyte transformation and chemiluminescence by spleen cells of normal and Cryp tococcus neoformans-infected mice. Infect. and Immun. 32 (1981) 1073-1078 9. Grosse, G., S. K. Mishra, and F. Staib: Selective involvement of the brain in experimen tal murine cryptococcosis. II. Histopathological observations. Zbl. Bakt. Hyg. 1. Abt. Orig. A 233 (1975) 106-122 10. Grosse, G., W. Heise und F. Staib: Hisroplasmose der Haut als opportunistische Erstin fektion bei AIDS. Dtsch. Med. Wschr. 118 (1993) 1555-1560 11. Howard, D. H.: Intracellular growth of Histoplasma capsulatum. J. Bacteriol. 89 (1965) 518-523 12. Howard, D. H.: Effect of temperature of intracellular growth of Histoplasma capsula tum. J. Bacterial. 93 (1967) 438-444 13. Howard, D. H., N. Dabrowa, V. Otto, and]. Rhodes: Cysteine transport and sulfite re ductase activity in a germinationdefective mutant of Histoplasma capsulatum. J. Bacte riol. 141 (1980) 417-421 14.Jensen, H. E., B. Bloch, P. Henriksen, H. H. Dietz, H. Schonheyder, and L. Kaufman: Disseminated histoplasmosis in a badger (Meles meles) in Denmark. APMIS 100 (1992) 586-592 15. Kaufman, L.: Immunohistologic diagnosis of systemic mycoses: an update. Eur. J. Epi demiol. 8 (1992) 377-382 16.Kwon-Chung, K.]. and J.E. Bennett: Medical Mycology. Lea & Febiger, Philadel phia-London (1992) 17. Medoff, G., G. S. Kobayashi, A. Painter, and S. Travis: Morphogenesis and pathogeni city of Histoplasma capsulatum. Infect. Immun. 55 (1987) 1355-1358 18. Priemer, J. and E. Lux: Helminths of badgers in Germany. Parassitologia (Roma) (Ab stracts) 38 (1996) 20 19. Rapp, J., A. Lofqvist, E. Breuer und H. Rang: SproGpilze als Ursache von Hautgranulo men beim Dachs in Siiddeutschland. Tierarztl. Umschau 47 (1992) 451-454 20. Rippon,]. w.: Medical Mycology. 3rd Ed., p. 405. W. B. Saunders Comp., Philadelphia (1988) 21. Robbins, St. L. and R. S. Cotran: Pathologic basis of disease. 2nd Ed., pp. 69-76. W. B. Saunders Comp., Philadelphia, London, Toronto (1979) 22. Scherr, G. H.: Studies on the dimorphism of Histoplasma capsulatum. 1. The roles of SH groups and incubation temperature. Exp. Cell Res. 12 (1957) 92-107
Histoplasmosis of man and badger
539
23 . Staib, F.: Cryptococcus neoformans and Cryptococcosis. Peculiarities and challenge: Opening lecture. J. Mycol. Med. 4 (1994) 56-60 24. Staib, F.: Bioabfall aus medizinisch-mykologischer Sicht. Bundesgesundheitsbl. 35 (1992) 21-26 25. Staib, F.: Editorial: Fungi in biowaste and compost. Medical-mycological aspects and proposals. Zbl. Bakt. 285 (1996) 1-4 26. Staib, F., S. K. Mishra, G. Grosse, and Th. Abel: Ocular cryptococcosis - experimental and clinical observations. Zbl. Bakt. Hyg. I. Abt. Orig. A 237 (1977) 378-394 27. Staib, F., W. Weller, S. Brem, R. Schindlmayr, and E. Schmittdiel: A Cryptococcus neo formans strain from the brain of a wildlife fox (Vulpes vulpes) suspected of rabies: My cological observations and comments. Zbl. Bakt. Hyg. A 260 (1985) 566-571 28. Staib, F. and G. Grosse: Brown-red pigment formation by the mycelial phase of a clini cal isolate of Histoplasma capsulatum on Staib agar. A preliminary report. Zbl. Bakt. 283 (1996)515-521 29. Summerbell, R. c., F. Staib, D. G. Ahearn et al.: Household hyphomycetes and other in door fungi. J. Med. Vet. Mycol. 32 (Suppl. 1) (1994) 277-286 30. Weller, w., S. Brem, R. Schindlmayr und E. Schmittdiel: Cryptococcose bei einem Rot fuchs (Vulpes vulpes). Berl. Munch. tierarztl. Wschr. 98 (1985) 14-15
Prof. Dr. med. G. Grosse, Institut fur Pathologie, Auguste-Viktoria-Krankenhaus, Ru bensstraRe 125, D-12157 Berlin, Germany Prof. Dr. med. Dr. F. Staib, BrentanostraRe 26, D-12163 Berlin, Germany
35
Zbl. Bakt 285/4