The Induction of Human Myospherulosis in Experimental Animals Katherine De Schryver-Kecskemeti, MD, and Michael Kyriakos, MD
Tissue lesions similar to those in human myospherulosis were reproduced in experimental animals and studied by light and electron microscopy. The lesions were produced by the use of petrolatum-based antibiotic ointments. These ointments, which are marketed as nonsterile products, are similar to those used for hemostatic packing in otolaryngologic surgery. To date, use of these ointments has been reported to precede the finding of human paranasal sinus myospherulosis. The exact nature of the structures of mvospherulosis remains unknown. The experimental evidence strongly suggests that they can be iatrogenically produced by the use of these nonsterile ointments. (Am J Pathol
87:33-40, 1977)
IN 1969, MCCLATCHIE AND ASSOCIATES described 7 patients from Kenya, East Africa, with peripheral soft tissue nodules which, histologically, contained spherical fungus-like structures. These structures were morphologically distinct from any of the known pathogenic fungi, and their exact nature could not be determined despite review by numerous specialists in mycology and parasitology. Due to the involvement of
skeletal muscle in their patients, they named this entity myospherulosis.1'2 Five patients were subsequently reported by Hutt et al. from Uganda in 1971.' The structures of myospherulosis do not take any of the usual stains for fungi, and attempts to grow them in vivo and in vitro have been unsuccessful. A series of 16 patients with myospherulosis was recently described from St. Louis, Missouri.' Unlike the African cases, the lesions were located in the paranasal sinuses, nose, and the middle ear. The histopathology of these lesions was similar to that described in the African cases. Briefly, the lesions were characterized by cystic tissue spaces of varying size which were lined by histiocytes and foreign body type giant cells. The surrounding stroma was fibrotic and chronically inflamed. Within tissue spaces measuring up to 1 mm large sporangium-like sacs (parent bodies) were seen which contained varying members of smaller endobodies (spherules). A distinctive feature of these structures was their dark brown coloration in hematoxylin and eosin-stained sections. Ultrastructurally, these spherules From the Department of Pathology, Division of Surgical Pathology, Washington Unihersity School of NMedicine, St. Louis, Missouri. Supported in part by Grant AO-5201 from the National Cancer Institute. Accepted for publication December 6, 1976. Address reprint requests to Dr. Katherine De Schryver-Kecskemeti, Division of Surgical Pathology, Barnes Hospital, St. Louis, MO 63110. 33
34
DE SCHRYVER-KECSKEMETI AND KYRIAKOS
American Journal of Pathology
had a thick, focally irregular electron-dense wall and an interior composed of unorganized granular material.' All of the St. Louis patients had a history of prior surgery in the areas subsequently found to contain the structures of myospherulosis. A chronic fibrogenic inflammatory response had necessitated further surgery in every case. A common denominator in these patients was the operative use of hemostatic packing containing either nonsterile petrolatum-based antibiotic ointments or gauze impregnated with petrolatum. Tetracycline was the most common antibiotic incorporated with the petrolatum. It was suggested that a relationship existed between the use of these nonsterile ointments and the finding of myospherulosis.4 To test this possibility, an attempt was made to reproduce the lesion by introducing the same materials used in humans into experimental animals and to isolate these structures directly from the ointments. Materials and Methods Animal Studies
Male Wistar rats weighing approximately 100 g were used in all experiments. The same type of tetracycline antibiotic ointment (3% Achromycin ointment, Lederle Laboratories) and sterile cotton gauze (Nu-Gauze, Johnson and Johnson, Co.) used during surgery in patients who subsequently developed myospherulosis was obtained from the Barnes Hospital pharmacy. The ointment consisted of a thick yellow paste supplied in 28.4-g tubes. Five tubes, each having different manufacturing lot numbers, were used throughout the experiments. Using several methods, the ointment and gauze was introduced into rats which had been placed under light ether anesthesia. Sterile technique was used throughout the experiments. The animals were divided into three groups; Group I contained 12 animals, Group II, 5; and Group III, 8. Group I
After shaving and preparing the area with absolute ethyl alcohol, we made a 2-cm cutaneous incision in the gluteal region. A pouch was formed in the subcutaneous tissue by blunt dissection, and the underlying gluteus muscle was crushed with a hemostatic clamp. Approximately 5 g of ointment was squeezed directly from the tube into the pouch, and the incision was closed with 3-0 sterile silk sutures. Group 11
After preparing the area, a midline abdominal skin incision was made and by blunt dissection a pouch was created in the subcutaneous tissues without entering the fascia of the rectus muscle. The muscle itself was not traumatized. The ointment was introduced and the incision closed as described above. Group Ill
Subsequent to shaving and preparing the abdomen, a midline abdominal incision was made and carried through into the peritoneal cavity. Animals in this group were further divided into Subgroups A, B, and C. In Subgroup A (4 animals), a 1 X 1 cm strip of sterile cotton gauze was implanted in the peritoneal cavity. In Subgroup B (2 animals), the sterile cotton gauze strip to which had been added about 5 g of the antibiotic ointment was
MYOSPHERULOSIS
Vol. 87, No. 1 April 1977
35
implanted; in Subgroup C (2 animals), the same amount of ointment alone was deposited into the abdominal cavity. When closing the incision, the peritoneal and cutaneous tissues were sutured separatelv. At time intervals varying from 3 to 47 days postoperatively, the animals were sacrificed with an overdose of ether (Table 1) and the operative sites were exicised in toto. Postmortem examination of each animal was done. Tissue samples for microscopic examinations were taken from any grossly apparent lesions; routine samples were taken from the lungs, liver, spleen, kidneys, and lymph nodes. The tissue was fixed in 10% neutral buffered formalin and was processed by the usual histologic techniques and stained with hematoxvlin and eosin. Tissue from the operative sites was examined using serial sections. The Grcott methenamine silver stain for fungi was used when indicated.' In addition, tissue from the operative sites was also processed for electron microscopy. Samples were fixed in 4% buffered glutaraldehyde and postfixed in 1 7c osmium tetroxide, after which they were dehydrated through a graded series of alcohols and processed routinely for electron microscopy. Using I-,g toluidine blue-stained sections, characteristic areas were selected for further ultrathin sectioning with a Huxley LKB microtome. Grids were double stained with uranyl acetate and lead citrate and examined in a Philips 300 electron microscope. Hnan Case Material Material from a patient with myospherulosis of the maxillary sinus and nose was used for comparison in the experimental animal study.5 Tissue fixed in buffered formalin and embedded in paraffin was reprocessed for electron microscopy. Areas which contained the most abundant and clearest view of the structures of myospherulosis were identified on the
hematoxylin and eosin-stained sections, and matched areas from the paraffin blocks were excised. After removal of the paraffin through several changes of xylene, the tissue was rehydrated through graded changes of ethyl alcohol. It was then placed in 0.15 NI phosphate buffer, pH 7.4, for 1 hour, and following postfixation in 2% osmium tetroxide for 30 minutes, tissue was processed in the usual manner for electron microscopy.
Isolaion Pro,edures To dissolve the petrolatum-based ointments and allow for microscopic examination, anhydrous ether, methylene chloride, xylene, and absolute ethyl alcohol were used as Table 1-Experimental Design
Group
No. of animals 12
5
11
8
111
Group A Group B
Site Subcutaneous gluteal region with crushing of muscle Subcutaneous abdominal wall
Product Tetracyline ointment Tetracyline ointment
Times of sacrifice (days) 3,5,8,11,21,34
7,11,19,21,34
Intraperitoneal Sterile cotton gauze (4) Sterile cotton
14
14,47
gauze with tetracyline ointment (2)
Group C
Tetracyline ointment (2)
14,21
DE SCHRYVER-KECSKEMETI AND KYRIAKOS
36
American Journal of Pathology
solvents. All the ointment that could be manually expressed from a single tube was placed in a large centrifuge tube. About 40 cu cm of one of the solvents was then layered onto the ointment, and the two were thoroughly mixed with a spatula. The mixture was then centrifuged at 3000 rev/min for 5 minutes. The resulting supernatant was decanted and saved. Any undissolved residue was resuspended in 40 ml of fresh solvent, mixed, and recentrifuged. Alcohol did not dissolve the ointment, but five washings with either of the other solvents proved adequate to dissolve all the ointment. The supernatants which appeared yellow were pooled, and under vacuum passed through a Millipore filter of 5 ,u pore size. The filter was then fixed with 95% ethyl alcohol and processed for routine cytology using the Papanicolaou technique. A total of six tubes of ointment was individually treated in this manner and examined. The tubes used had different lot numbers and were different from the ones used in the animal studies.
Results
During the course of the experiments, the animals exhibited no ill effects from their treatment. The operative sites were almost entirely healed by the second postoperative week. Gross Pathology
The macroscopic appearance of the lesions was identical in animals of Groups I and II. The tissue reaction was found to be limited to the area of the ointment deposition. In 3- and 5-day-old lesions, a "pocket" of fibrous tissue had formed around the operative site. Within this pocket, serous fluid had accumulated that contained yellowish particulate matter. At 8 days, the serous fluid was no longer present, no yellow particulate matter could be identified, and the subcutaneous fascia was focally thickened, with areas of a fine bubbly appearance. At later time intervals, the gross appearance remained essentially the same except for an increasing fibrous reaction around the operative site. In Group III animals, the gross findings differed in the three subgroups. In Subgroup A, the cotton gauze was found to be walled off between the abdominal wall and the right colon by fibrous tissue. No gross abnormality of the intestinal serosa, mesothelium, or abdominal organs was seen. In the 2 animals of Subgroup B, the cotton gauze was similarly walled off between the abdominal wall and the right colon, but in this group there were patchy areas of thickening of the capsules of the liver, spleen, and kidneys, with a fine bubbly appearance similar to that seen on the fascia of Groups I and II. The underlying organ parenchyma, however, appeared normal, and there was no fluid accumulation in the peritoneal cavity. The gross findings in both animals of Subgroup C were similar to those in Subgroup B except for the absence of the cotton gauze. Light Microscopy
Sections of the material from animals in Groups I and II showed a
Vol. 87, No. 1 April 1977
MYOSPHERULOSIS
37
similar histologic pattern. At 3 and 5 days, there was an accumulation of an amorphous material within the soft tissue of the operative sites. This material was highly eosinophilic and contained a crystalline needle-like component (Figures 1 and 2). It was not seen in lesions older than 11 days. A fibrinous exudate with an acute inflammatory reaction was also present. At 8 and 11 days, a chronic inflammatory infiltrate was present at the operative site consisting of lymphocytes and phagocytic cells. In addition, closely arranged tissue spaces or holes varying in size from several microns to over a millimeter in diameter were now present (Figure 2A). They corresponded to the "bubbly" areas seen macroscopically. These spaces were lined either by a condensation of the surrounding fibrous tissue or by histiocytes and multinucleate foreign body type giant cells. Some tissue spaces were empty, while others contained varying amounts of highly eosinophilic debris (Figures 2A and 3). This debris began to show a distinct brown coloration beginning at Day 14. Some tissue spaces also contained scattered oval, sac-like bodies which measured 20 and 50 ;i in diameter, and which were limited by a distinct wall. These structures or "parent bodies" contained varying numbers of closely packed endobodies, or "'spherules" (Figure 4A-C). These spherules measured 5 to 7 i in diameter, had an irregular contour with a sharply demarcated outer wall, and appeared to lack any internal structure. Both the parent bodies and the enclosed spherules failed to take the Grocott stain. Their morphology was very similar to that found in human myospherulosis (Figures 4D-F and 5). At Day 11, a fully developed tissue lesion indistinguishable from that of the human case material was present in the gluteal muscle (Figure 6A and B). The lesions did not extend beyond the original operative area. Those in Group I animals involved only the superficial portions of the gluteus muscle in spite of the added mechanical crush injury. In Group II, where the fascia had been left intact, the lesion was confined to the subcutaneous tissue. In animals in Group IIIA, the implanted sterile cotton gauze showed only foreign body giant cell reaction with fibrosis in the surrounding soft tissue. Tissue spaces, parent bodies, spherules, and eosinophilic debris were not seen. In Group IIIB animals, there was foreign body giant cell reaction around the implanted cotton gauze, and all the histologic features described in Groups I and II were seen. The eosinophilic parent bodies and spherules were present up to 47 days in this material. Group IIIC animals also showed essentially the same histopathology as the animals in Groups I and II. Focal lesions consisting of closely arranged tissue spaces containing eosinophilic debris were found on the surface of
38
DE SCHRYVER-KECSKEMETI AND KYRIAKOS
American Journal of Pathology
the peritoneum, and on the capsules of the liver, kidney, and spleen, without parenchymal involvement. The lungs and lymph nodes did not show any involvement. Electron Microscopy
Electron microscopy of all the petrolatum-induced lesions showed similar features regardless of the operative site used. In 3-, 5-, and 8-day-old lesions, phagocytic cells rich in cytoplasmic organelles and with numerous microvilli were present. The cytoplasm of these cells contained engulfed electron-dense debris and lipid vacuoles (Figure 7A and B). This same electron-dense material was also seen lying free outside cells in the 3-dayold lesions (Figure 8A). Among the debris, round to oval bodies were seen which measured 4 to 5 ,u in diameter. They had what appeared to be a double electron-dense outer wall, the thickness of which varied between 400 and 800 A. The interior of these structures contained only amorphous granular material. Occasional openings or discontinuities were seen on both inner and outer aspects of their walls (Figure 8A). The outer wall had in addition a coarse irregular outline caused by aggregates or clumps of electron-dense material. These bodies were similar in size, shape, and staining characteristics to the spherules present in human material (Figure 8B). Isolation Results
Examination of the Millipore filters showed only the presence of eosinophilic crystalline material similar to that seen in the tissues at Day 3 and 5. No structures similar to those found in the tissue were recovered. Discussion In these experiments, a tissue reaction similar to that seen in human cases of myospherulosis 1-4 was found in every instance in which the petrolatum-based ointment was used. In addition, the basic components of the disease, namely parent bodies and spherules, were also regularly found within the tissues. There were, however, qualitative differences between the experimental and human disease. In tissue from the experimental animals, the parent bodies were smaller, less abundant, and enclosed fewer endobodies than those in the human material. However, even in the human material there has been wide variability from case to case (and even from section to section) in the concentration and size of
these structures.4 Several factors could be involved in determining the tissue concentration of these structures. There is no conclusive evidence that we are
Vol. 87, No. 1 April 1977
MYOSPHERULOSIS
39
dealing with an organism capable of reproduction. If this material is indeed some form of inert vegetable matter representing exogenous contamination of the ointment during its manufacture, then the amount of material in the tissue would be related to both its concentration within the ointment, the quantity of ointment introduced, and the ability of the animals to clear the foreign matter from the tissue. Significant in this regard is that these ointments are marketed nonsterile products for external use, and as such, their quality control is not governed by Federal regulations.7 However, if these structures are organisms capable of reproduction, then in addition to the aforementioned factors, conditions which influence the ability of the organism to survive (such as anatomic site, tissue temperature, and local oxygen tension) would be determinants of the concentration of these structures in the tissue. The color of the debris, parent bodies, and spherules in the St. Louis cases was reported as being a distinctly brown to brownish-black, with a pale eosinophilia present only in structures found in areas of tissue necrosis. In the African material, the structures of myospherulosis were uniformly palely eosinophilic. In the experimental material, distinctly brown parent bodies and spherules were found in lesions after 14 days. However, structures with a highly eosinophilic color were still seen even in animals sacrificed at 47 days. The significance of the pigmentation in these structures is unknown. The fine structure of the spherules in the experimental lesion was essentially the same as those described in material from a single human case.' However, minor ultrastructural differences exist between the human and experimental material. In the human material the spherules showed duplication of their walls only rarely, with variation in wall thickness and irregularities on their inner surfaces caused by electrondense clumps. Pores or openings were also present in the walls of all spherules found outside the parent bodies. Unfortunately, in the experimental material, no parent body was seen in the preparations for electron microscopy which may or may not be due to sampling problems. The spherules always showed duplication of their walls over a relatively large portion of their circumference. The outer wall contained focal irregularities due to electron-dense clumps with focal discontinuities or holes seen in both wall components. They appeared different from the pores noted in the human material. Some of these morphologic differences might well be related to the fact that the tissues from the single human case had been formalin fixed and paraffin embedded prior to reprocessing for electron microscopy.5 In these experiments, different tissue sites were chosen in an attempt to
40
DE SCHRYVER-KECSKEMETI AND KYRIAKOS
American Journal of Pathology
enhance the possibility of lesion development. Since originally described from the peripheral soft tissues, the operative sites used in Groups I and II were chosen to imitate these conditions. Additional mechanical crushing of the gluteal muscle in Group I was an attempt to mimic further operative trauma. The intraperitoneal site used in Group III animals was chosen as one which might favor the growth of an "organism." In spite of this design, all lesions appeared identical whatever the site used, and the lesions did not spread beyond the local area. The human disease has also been confined to local areas without systemic involvement.4 The ointment used in these animals contains a petrolatum base as a vehicle for the tetracycline antibiotic. In humans, while a variety of ointments were used, they all contained a petrolatum base. Since we were able to reproduce a lesion similar to that seen in humans by using this material, the obvious inference is that the ointment itself contains the structures of myospherulosis. The most direct and convincing evidence for this would be the actual isolation of these bodies from the ointment. Unfortunately, with the crude solvent procedures which were used, we could not recover any material which resembled the structures of myospherulosis. Some of the solvents used in the experiments are known not to affect or dissolve the structures of myospherulosis, since tissues containing these are passed through numerous changes of xylene and alcohol during their processing for histology. The lack of recovery of these structures on the Millipore filters thus cannot be explained on these grounds. Interaction of the material with viable tissues might be a requirement for their development. Until the nature of these structures is further clarified, the proposition that the ointments are the source of this material must remain tentative. References 1.
2. 3. 4.
5. 6.
7.
McClatchie S, Warambo MW, Bremner AD: Myospherulosis: A previously unreported disease? Am J Clin Pathol 51:699-704, 1969 McClatchie S, Bremner AD: Unusual subcutaneous swellings in African patients. E Afr Med J 46:625-633, 1969 Hutt MSR, Fernandes BJJ, Templeton AC: Myospherulosis: Subcutaneous spherulocystic disease. Trans R Soc Trop Med Hyg 65:182-188, 1971 Kyriakos M: Myospherulosis of the paranasal sinuses, nose and middle ear: A possible iatrogenic disease. Am J Clin Pathol (In press) De Schryver-Kecskemeti K, Kyriakos M: Myospherulosis: An electron microscopic study of a human case. Am J Clin Pathol (In press) Lillie RD: Histopathologic Technique and Practical Histochemistry, Third edition. New York, McGraw Hill Book Co., 1965 Code of Federal Regulations: Title 21-Foods and Drugs. Office of the Federal Register, National Archives and Record Service, General Services Administration. US Printing Office, Washington, DC, Revised April, 1975
MYOSPHERULOSIS
Vol. 87, No.1 April 1977
[Illustrations follow]
41
Legends for Figures Figure 1-Three-day lesion of the gluteal region with acute fibrinous exudate in which crystalline foreign material is seen (arrow) (H&E, x 90).
Figure 2A-Eleven-day lesion of the gluteal region. Tissue spaces are seen containing debris in a well-vascularized connective tissue stroma. (H&E, x 90) B-Higher power of area shown by arrow in A. Crystalline-like material is seen surrounded by a cuff of chronic inflammatory cells. (H&E, x 600)
Figure 3-Section of grossly "bubbly" material seen at Day 21 in the abdominal rectus fascia.
Nodular array of multicystic spaces, some containing debris, are surrounded by mild chronic inflammatory infiltrate and foreign body giant cells. Edematous stroma separates it from underlying rectus muscle seen in the lower part of the figure. (H&E, x 150)
1~~~~~~~~~~~~~~~~:1
:ft.41.~~~~~~~~~~~~~~~~~~~~~~~~~-10--
-
t
-A
..~~~~~~-Sx,
i:.
..
I
Figures 4A-C-Structures seen within tissue spaces in experimental animals (H&E, x 720). D-F-Structures of myospherulosis from human material (H&E, x 720).
Figure 5-Two large parent bodies with their enclosed endospores from a case of human myospherulosis. One parent body has an intact wall, while the other appears to be focally disrupted. (H&E, x 720) -AL
.e
?'.
ii, 1.
Cw
;..c
iIV
..
All.
Fgum 6A-Elven-day experimental lesion in animal from Group I shows multipe tssue spaces, chronic inflammation, and cellular debris, bearing striking similarity to human eion shown in B (H&E, x 90). B-Maxillary sinus tissue of patient with myospheruosis. Note tissue spaces containing dark debris. (H&E, x 90)
f
.,
.'+.;%i
TO
.,
.
r :.
41.,.
'i
"'
'A
Figures 7A and B-Phagocytic cells showing multiple cytoplasmic microvilli. Engulfed electron-dense foreign
material (long arrows) and fat vacuoles (arrow head) are seen within the cytoplasm. (Uranyl acetate and lead citrate)
Fe
a
l
3
o
-
f
(arro)ewcnt Il bodies sittsh
mti
eohaiuesnidttfld
J%~~~~~~~~~~S
wals Discniute an
ireuarte ar see
myophruss PorMaesiaarem evidentol leionthec material. (Uranyl acetate and lead citrate )
in th wal.Th intro conist of unrgaie
gaur
gatrianla neircnitfuoreganie walfh electron-dense hwcteexrclua
Tissue lesions similar to those in human myospherulosis were reproduced in experimental animals and studied by light and electron microscopy. The lesions were produced by the use of petrolatum-based antibiotic ointments. These ointments, which are marketed as nonsterile products, are similar to those used for hemostatic packing in otolaryngologic surgery. To date, use of these ointments has been reported to precede the finding of human paranasal sinus myospherulosis. The exact nature of the structures of mvospherulosis remains unknown. The experimental evidence strongly suggests that they can be iatrogenically produced by the use of these nonsterile ointments. (Am J Pathol
87:33-40, 1977)
IN 1969, MCCLATCHIE AND ASSOCIATES described 7 patients from Kenya, East Africa, with peripheral soft tissue nodules which, histologically, contained spherical fungus-like structures. These structures were morphologically distinct from any of the known pathogenic fungi, and their exact nature could not be determined despite review by numerous specialists in mycology and parasitology. Due to the involvement of
skeletal muscle in their patients, they named this entity myospherulosis.1'2 Five patients were subsequently reported by Hutt et al. from Uganda in 1971.' The structures of myospherulosis do not take any of the usual stains for fungi, and attempts to grow them in vivo and in vitro have been unsuccessful. A series of 16 patients with myospherulosis was recently described from St. Louis, Missouri.' Unlike the African cases, the lesions were located in the paranasal sinuses, nose, and the middle ear. The histopathology of these lesions was similar to that described in the African cases. Briefly, the lesions were characterized by cystic tissue spaces of varying size which were lined by histiocytes and foreign body type giant cells. The surrounding stroma was fibrotic and chronically inflamed. Within tissue spaces measuring up to 1 mm large sporangium-like sacs (parent bodies) were seen which contained varying members of smaller endobodies (spherules). A distinctive feature of these structures was their dark brown coloration in hematoxylin and eosin-stained sections. Ultrastructurally, these spherules From the Department of Pathology, Division of Surgical Pathology, Washington Unihersity School of NMedicine, St. Louis, Missouri. Supported in part by Grant AO-5201 from the National Cancer Institute. Accepted for publication December 6, 1976. Address reprint requests to Dr. Katherine De Schryver-Kecskemeti, Division of Surgical Pathology, Barnes Hospital, St. Louis, MO 63110. 33
34
DE SCHRYVER-KECSKEMETI AND KYRIAKOS
American Journal of Pathology
had a thick, focally irregular electron-dense wall and an interior composed of unorganized granular material.' All of the St. Louis patients had a history of prior surgery in the areas subsequently found to contain the structures of myospherulosis. A chronic fibrogenic inflammatory response had necessitated further surgery in every case. A common denominator in these patients was the operative use of hemostatic packing containing either nonsterile petrolatum-based antibiotic ointments or gauze impregnated with petrolatum. Tetracycline was the most common antibiotic incorporated with the petrolatum. It was suggested that a relationship existed between the use of these nonsterile ointments and the finding of myospherulosis.4 To test this possibility, an attempt was made to reproduce the lesion by introducing the same materials used in humans into experimental animals and to isolate these structures directly from the ointments. Materials and Methods Animal Studies
Male Wistar rats weighing approximately 100 g were used in all experiments. The same type of tetracycline antibiotic ointment (3% Achromycin ointment, Lederle Laboratories) and sterile cotton gauze (Nu-Gauze, Johnson and Johnson, Co.) used during surgery in patients who subsequently developed myospherulosis was obtained from the Barnes Hospital pharmacy. The ointment consisted of a thick yellow paste supplied in 28.4-g tubes. Five tubes, each having different manufacturing lot numbers, were used throughout the experiments. Using several methods, the ointment and gauze was introduced into rats which had been placed under light ether anesthesia. Sterile technique was used throughout the experiments. The animals were divided into three groups; Group I contained 12 animals, Group II, 5; and Group III, 8. Group I
After shaving and preparing the area with absolute ethyl alcohol, we made a 2-cm cutaneous incision in the gluteal region. A pouch was formed in the subcutaneous tissue by blunt dissection, and the underlying gluteus muscle was crushed with a hemostatic clamp. Approximately 5 g of ointment was squeezed directly from the tube into the pouch, and the incision was closed with 3-0 sterile silk sutures. Group 11
After preparing the area, a midline abdominal skin incision was made and by blunt dissection a pouch was created in the subcutaneous tissues without entering the fascia of the rectus muscle. The muscle itself was not traumatized. The ointment was introduced and the incision closed as described above. Group Ill
Subsequent to shaving and preparing the abdomen, a midline abdominal incision was made and carried through into the peritoneal cavity. Animals in this group were further divided into Subgroups A, B, and C. In Subgroup A (4 animals), a 1 X 1 cm strip of sterile cotton gauze was implanted in the peritoneal cavity. In Subgroup B (2 animals), the sterile cotton gauze strip to which had been added about 5 g of the antibiotic ointment was
MYOSPHERULOSIS
Vol. 87, No. 1 April 1977
35
implanted; in Subgroup C (2 animals), the same amount of ointment alone was deposited into the abdominal cavity. When closing the incision, the peritoneal and cutaneous tissues were sutured separatelv. At time intervals varying from 3 to 47 days postoperatively, the animals were sacrificed with an overdose of ether (Table 1) and the operative sites were exicised in toto. Postmortem examination of each animal was done. Tissue samples for microscopic examinations were taken from any grossly apparent lesions; routine samples were taken from the lungs, liver, spleen, kidneys, and lymph nodes. The tissue was fixed in 10% neutral buffered formalin and was processed by the usual histologic techniques and stained with hematoxvlin and eosin. Tissue from the operative sites was examined using serial sections. The Grcott methenamine silver stain for fungi was used when indicated.' In addition, tissue from the operative sites was also processed for electron microscopy. Samples were fixed in 4% buffered glutaraldehyde and postfixed in 1 7c osmium tetroxide, after which they were dehydrated through a graded series of alcohols and processed routinely for electron microscopy. Using I-,g toluidine blue-stained sections, characteristic areas were selected for further ultrathin sectioning with a Huxley LKB microtome. Grids were double stained with uranyl acetate and lead citrate and examined in a Philips 300 electron microscope. Hnan Case Material Material from a patient with myospherulosis of the maxillary sinus and nose was used for comparison in the experimental animal study.5 Tissue fixed in buffered formalin and embedded in paraffin was reprocessed for electron microscopy. Areas which contained the most abundant and clearest view of the structures of myospherulosis were identified on the
hematoxylin and eosin-stained sections, and matched areas from the paraffin blocks were excised. After removal of the paraffin through several changes of xylene, the tissue was rehydrated through graded changes of ethyl alcohol. It was then placed in 0.15 NI phosphate buffer, pH 7.4, for 1 hour, and following postfixation in 2% osmium tetroxide for 30 minutes, tissue was processed in the usual manner for electron microscopy.
Isolaion Pro,edures To dissolve the petrolatum-based ointments and allow for microscopic examination, anhydrous ether, methylene chloride, xylene, and absolute ethyl alcohol were used as Table 1-Experimental Design
Group
No. of animals 12
5
11
8
111
Group A Group B
Site Subcutaneous gluteal region with crushing of muscle Subcutaneous abdominal wall
Product Tetracyline ointment Tetracyline ointment
Times of sacrifice (days) 3,5,8,11,21,34
7,11,19,21,34
Intraperitoneal Sterile cotton gauze (4) Sterile cotton
14
14,47
gauze with tetracyline ointment (2)
Group C
Tetracyline ointment (2)
14,21
DE SCHRYVER-KECSKEMETI AND KYRIAKOS
36
American Journal of Pathology
solvents. All the ointment that could be manually expressed from a single tube was placed in a large centrifuge tube. About 40 cu cm of one of the solvents was then layered onto the ointment, and the two were thoroughly mixed with a spatula. The mixture was then centrifuged at 3000 rev/min for 5 minutes. The resulting supernatant was decanted and saved. Any undissolved residue was resuspended in 40 ml of fresh solvent, mixed, and recentrifuged. Alcohol did not dissolve the ointment, but five washings with either of the other solvents proved adequate to dissolve all the ointment. The supernatants which appeared yellow were pooled, and under vacuum passed through a Millipore filter of 5 ,u pore size. The filter was then fixed with 95% ethyl alcohol and processed for routine cytology using the Papanicolaou technique. A total of six tubes of ointment was individually treated in this manner and examined. The tubes used had different lot numbers and were different from the ones used in the animal studies.
Results
During the course of the experiments, the animals exhibited no ill effects from their treatment. The operative sites were almost entirely healed by the second postoperative week. Gross Pathology
The macroscopic appearance of the lesions was identical in animals of Groups I and II. The tissue reaction was found to be limited to the area of the ointment deposition. In 3- and 5-day-old lesions, a "pocket" of fibrous tissue had formed around the operative site. Within this pocket, serous fluid had accumulated that contained yellowish particulate matter. At 8 days, the serous fluid was no longer present, no yellow particulate matter could be identified, and the subcutaneous fascia was focally thickened, with areas of a fine bubbly appearance. At later time intervals, the gross appearance remained essentially the same except for an increasing fibrous reaction around the operative site. In Group III animals, the gross findings differed in the three subgroups. In Subgroup A, the cotton gauze was found to be walled off between the abdominal wall and the right colon by fibrous tissue. No gross abnormality of the intestinal serosa, mesothelium, or abdominal organs was seen. In the 2 animals of Subgroup B, the cotton gauze was similarly walled off between the abdominal wall and the right colon, but in this group there were patchy areas of thickening of the capsules of the liver, spleen, and kidneys, with a fine bubbly appearance similar to that seen on the fascia of Groups I and II. The underlying organ parenchyma, however, appeared normal, and there was no fluid accumulation in the peritoneal cavity. The gross findings in both animals of Subgroup C were similar to those in Subgroup B except for the absence of the cotton gauze. Light Microscopy
Sections of the material from animals in Groups I and II showed a
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similar histologic pattern. At 3 and 5 days, there was an accumulation of an amorphous material within the soft tissue of the operative sites. This material was highly eosinophilic and contained a crystalline needle-like component (Figures 1 and 2). It was not seen in lesions older than 11 days. A fibrinous exudate with an acute inflammatory reaction was also present. At 8 and 11 days, a chronic inflammatory infiltrate was present at the operative site consisting of lymphocytes and phagocytic cells. In addition, closely arranged tissue spaces or holes varying in size from several microns to over a millimeter in diameter were now present (Figure 2A). They corresponded to the "bubbly" areas seen macroscopically. These spaces were lined either by a condensation of the surrounding fibrous tissue or by histiocytes and multinucleate foreign body type giant cells. Some tissue spaces were empty, while others contained varying amounts of highly eosinophilic debris (Figures 2A and 3). This debris began to show a distinct brown coloration beginning at Day 14. Some tissue spaces also contained scattered oval, sac-like bodies which measured 20 and 50 ;i in diameter, and which were limited by a distinct wall. These structures or "parent bodies" contained varying numbers of closely packed endobodies, or "'spherules" (Figure 4A-C). These spherules measured 5 to 7 i in diameter, had an irregular contour with a sharply demarcated outer wall, and appeared to lack any internal structure. Both the parent bodies and the enclosed spherules failed to take the Grocott stain. Their morphology was very similar to that found in human myospherulosis (Figures 4D-F and 5). At Day 11, a fully developed tissue lesion indistinguishable from that of the human case material was present in the gluteal muscle (Figure 6A and B). The lesions did not extend beyond the original operative area. Those in Group I animals involved only the superficial portions of the gluteus muscle in spite of the added mechanical crush injury. In Group II, where the fascia had been left intact, the lesion was confined to the subcutaneous tissue. In animals in Group IIIA, the implanted sterile cotton gauze showed only foreign body giant cell reaction with fibrosis in the surrounding soft tissue. Tissue spaces, parent bodies, spherules, and eosinophilic debris were not seen. In Group IIIB animals, there was foreign body giant cell reaction around the implanted cotton gauze, and all the histologic features described in Groups I and II were seen. The eosinophilic parent bodies and spherules were present up to 47 days in this material. Group IIIC animals also showed essentially the same histopathology as the animals in Groups I and II. Focal lesions consisting of closely arranged tissue spaces containing eosinophilic debris were found on the surface of
38
DE SCHRYVER-KECSKEMETI AND KYRIAKOS
American Journal of Pathology
the peritoneum, and on the capsules of the liver, kidney, and spleen, without parenchymal involvement. The lungs and lymph nodes did not show any involvement. Electron Microscopy
Electron microscopy of all the petrolatum-induced lesions showed similar features regardless of the operative site used. In 3-, 5-, and 8-day-old lesions, phagocytic cells rich in cytoplasmic organelles and with numerous microvilli were present. The cytoplasm of these cells contained engulfed electron-dense debris and lipid vacuoles (Figure 7A and B). This same electron-dense material was also seen lying free outside cells in the 3-dayold lesions (Figure 8A). Among the debris, round to oval bodies were seen which measured 4 to 5 ,u in diameter. They had what appeared to be a double electron-dense outer wall, the thickness of which varied between 400 and 800 A. The interior of these structures contained only amorphous granular material. Occasional openings or discontinuities were seen on both inner and outer aspects of their walls (Figure 8A). The outer wall had in addition a coarse irregular outline caused by aggregates or clumps of electron-dense material. These bodies were similar in size, shape, and staining characteristics to the spherules present in human material (Figure 8B). Isolation Results
Examination of the Millipore filters showed only the presence of eosinophilic crystalline material similar to that seen in the tissues at Day 3 and 5. No structures similar to those found in the tissue were recovered. Discussion In these experiments, a tissue reaction similar to that seen in human cases of myospherulosis 1-4 was found in every instance in which the petrolatum-based ointment was used. In addition, the basic components of the disease, namely parent bodies and spherules, were also regularly found within the tissues. There were, however, qualitative differences between the experimental and human disease. In tissue from the experimental animals, the parent bodies were smaller, less abundant, and enclosed fewer endobodies than those in the human material. However, even in the human material there has been wide variability from case to case (and even from section to section) in the concentration and size of
these structures.4 Several factors could be involved in determining the tissue concentration of these structures. There is no conclusive evidence that we are
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dealing with an organism capable of reproduction. If this material is indeed some form of inert vegetable matter representing exogenous contamination of the ointment during its manufacture, then the amount of material in the tissue would be related to both its concentration within the ointment, the quantity of ointment introduced, and the ability of the animals to clear the foreign matter from the tissue. Significant in this regard is that these ointments are marketed nonsterile products for external use, and as such, their quality control is not governed by Federal regulations.7 However, if these structures are organisms capable of reproduction, then in addition to the aforementioned factors, conditions which influence the ability of the organism to survive (such as anatomic site, tissue temperature, and local oxygen tension) would be determinants of the concentration of these structures in the tissue. The color of the debris, parent bodies, and spherules in the St. Louis cases was reported as being a distinctly brown to brownish-black, with a pale eosinophilia present only in structures found in areas of tissue necrosis. In the African material, the structures of myospherulosis were uniformly palely eosinophilic. In the experimental material, distinctly brown parent bodies and spherules were found in lesions after 14 days. However, structures with a highly eosinophilic color were still seen even in animals sacrificed at 47 days. The significance of the pigmentation in these structures is unknown. The fine structure of the spherules in the experimental lesion was essentially the same as those described in material from a single human case.' However, minor ultrastructural differences exist between the human and experimental material. In the human material the spherules showed duplication of their walls only rarely, with variation in wall thickness and irregularities on their inner surfaces caused by electrondense clumps. Pores or openings were also present in the walls of all spherules found outside the parent bodies. Unfortunately, in the experimental material, no parent body was seen in the preparations for electron microscopy which may or may not be due to sampling problems. The spherules always showed duplication of their walls over a relatively large portion of their circumference. The outer wall contained focal irregularities due to electron-dense clumps with focal discontinuities or holes seen in both wall components. They appeared different from the pores noted in the human material. Some of these morphologic differences might well be related to the fact that the tissues from the single human case had been formalin fixed and paraffin embedded prior to reprocessing for electron microscopy.5 In these experiments, different tissue sites were chosen in an attempt to
40
DE SCHRYVER-KECSKEMETI AND KYRIAKOS
American Journal of Pathology
enhance the possibility of lesion development. Since originally described from the peripheral soft tissues, the operative sites used in Groups I and II were chosen to imitate these conditions. Additional mechanical crushing of the gluteal muscle in Group I was an attempt to mimic further operative trauma. The intraperitoneal site used in Group III animals was chosen as one which might favor the growth of an "organism." In spite of this design, all lesions appeared identical whatever the site used, and the lesions did not spread beyond the local area. The human disease has also been confined to local areas without systemic involvement.4 The ointment used in these animals contains a petrolatum base as a vehicle for the tetracycline antibiotic. In humans, while a variety of ointments were used, they all contained a petrolatum base. Since we were able to reproduce a lesion similar to that seen in humans by using this material, the obvious inference is that the ointment itself contains the structures of myospherulosis. The most direct and convincing evidence for this would be the actual isolation of these bodies from the ointment. Unfortunately, with the crude solvent procedures which were used, we could not recover any material which resembled the structures of myospherulosis. Some of the solvents used in the experiments are known not to affect or dissolve the structures of myospherulosis, since tissues containing these are passed through numerous changes of xylene and alcohol during their processing for histology. The lack of recovery of these structures on the Millipore filters thus cannot be explained on these grounds. Interaction of the material with viable tissues might be a requirement for their development. Until the nature of these structures is further clarified, the proposition that the ointments are the source of this material must remain tentative. References 1.
2. 3. 4.
5. 6.
7.
McClatchie S, Warambo MW, Bremner AD: Myospherulosis: A previously unreported disease? Am J Clin Pathol 51:699-704, 1969 McClatchie S, Bremner AD: Unusual subcutaneous swellings in African patients. E Afr Med J 46:625-633, 1969 Hutt MSR, Fernandes BJJ, Templeton AC: Myospherulosis: Subcutaneous spherulocystic disease. Trans R Soc Trop Med Hyg 65:182-188, 1971 Kyriakos M: Myospherulosis of the paranasal sinuses, nose and middle ear: A possible iatrogenic disease. Am J Clin Pathol (In press) De Schryver-Kecskemeti K, Kyriakos M: Myospherulosis: An electron microscopic study of a human case. Am J Clin Pathol (In press) Lillie RD: Histopathologic Technique and Practical Histochemistry, Third edition. New York, McGraw Hill Book Co., 1965 Code of Federal Regulations: Title 21-Foods and Drugs. Office of the Federal Register, National Archives and Record Service, General Services Administration. US Printing Office, Washington, DC, Revised April, 1975
MYOSPHERULOSIS
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[Illustrations follow]
41
Legends for Figures Figure 1-Three-day lesion of the gluteal region with acute fibrinous exudate in which crystalline foreign material is seen (arrow) (H&E, x 90).
Figure 2A-Eleven-day lesion of the gluteal region. Tissue spaces are seen containing debris in a well-vascularized connective tissue stroma. (H&E, x 90) B-Higher power of area shown by arrow in A. Crystalline-like material is seen surrounded by a cuff of chronic inflammatory cells. (H&E, x 600)
Figure 3-Section of grossly "bubbly" material seen at Day 21 in the abdominal rectus fascia.
Nodular array of multicystic spaces, some containing debris, are surrounded by mild chronic inflammatory infiltrate and foreign body giant cells. Edematous stroma separates it from underlying rectus muscle seen in the lower part of the figure. (H&E, x 150)
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Figure 5-Two large parent bodies with their enclosed endospores from a case of human myospherulosis. One parent body has an intact wall, while the other appears to be focally disrupted. (H&E, x 720) -AL
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