Henry Ford Hosp Med Journal Vol 27, No 2, 1979

Isolation of an Acid-Fast Organism from the Aqueous Humor in a Case of Sarcoidosis

C. L. Barth, PhD,* M. S. Judge, MS,** L. H. Mattman, PhD,** and P. C. Hessburg, MD * * *

The anterior chamber fluid from the eye of this patient with Introduction sarcoidosis was found to contain microcolonies of a cell- wall-deficient organism that was propagated and identified as acid-fast by the Intensified Acid-Fast stain. The colonies Anterior uveitis is frequently a local manifestation of were inoculated into mice and retrieved from the dead or systemic Boeck's sarcoidosis.^-^ In fact, an early name for sacrificed animals. This report suggests that the acid-fast sarcoidosis was uveoparotid fever. In such cases the aque­ microbe in the aqueous humor of this case of uveitis- ous humor appears to be sterile by routine culture methods. sarcoidosis may be the same organism as that found in the Similarly, in the case described here no classical microorga­ blood in sarcoidosis. Thus, it may be associated not only nisms appeared in a battery of media for aerobes, an­ with the primary disease, but also with the complications of aerobes. Mycoplasma, Mycobacteria, and fungi. However, Boeck's sarcoidosis. special media and staining detected an organism which resembles the isolates from the blood of nine other cases of Boeck's sarcoidosis evaluated in a parallel study.^ Data regarding four ofthese patients have been published.^ This acid-fast organism remains to be identified, but wfth im­ proved staining and culture methods diagnosis of sar- coidosis-and its complications may be facilitated.

Case Report A 32-year-old black man presented at the Ophthalmology Clinic with an inftamed left eye. A tissue diagnosis of sarcoid had been made following lung surgery six months before his eye difficulties started. The left eye contained many large keratitic precipitates on the cornea, an active aqueous flare, many posterior synechia (iris lens adhesions), cataract, and an elevated intraocular pressure. A diagnosis was made of granulomatous uveitis secondary to sarcoid with secondary glaucoma. In the next four months intraocular inftammation continued without relief despite the use of local, systemic and retrobulbar steroids. Treatment also required two glaucoma operations and the extraction of secondary cataract. The first tap (paracentesis) ofthe anterior chamber was performed four months after the onset of inflammation using the technique of Goldman and Cirard."'^ Inflammation continued for three months after the cataract was removed despite the use of steroids. Since a cell-wall-deflcient infection was suspected, erythromycin (250 mg four times daily) and tri-sulfapyrimidine (500 mg three times daily) were given orally. Each was used during alternating weeks for six Submitted for publication; October 2, 1978 weeks and then discontinued. The eye remained quiet for ten Accepted for publication; February 19,1979 months after antibiotics were stopped. At that time the anterior * Department of Pathology, Henry Ford Hospital uveitis recurred and a second paracentesis was performed on the ** Department of Biology, Wayne State University, Detroit, Michigan left eye. Cell-wall-deficient forms were isolated only from cultures *** Detroit Institute of Ophthalmology, Grosse Pointe Park, Michigan of the anterior chamber fluid taken during the flrst paracentesis. Address reprint requests to Dr. Barth, Departmentof Pathology, Henry Ford This report concerns on ly those organisms propagated from the flrst Hospital, 2799 W Grand Blvd, Detroit, Ml 48202 sample of aqueous humor.

127 Isolation of Acid-fast Organism from Aqueous Humor

Special Laboratory Studies Growth occurred only at 37°C rather than at 25°C and was Microdrops (0.03 ml) of anterior chamber fluid were treated minimal under anaerobic conditions. Hypertonic medium as described in a previousreport.^'^AIiquotsof growth inthe containing 10% sucrose did not improve growth. Medill-O'Kane Broth were frozen and later subcultured to Animal inoculation Kirschner's broth and Horse Muscle broth^ for animal inoculation. Six mice were inoculated intraperitoneally Four mice given the acid-fast culture and a cortisone dosage with cuftures of the acid-fast organism and were simul­ of 50 mg died between eight and eleven days. Two mice taneously given cortisone subcutaneously. In addftion to given 40 mg of cortisone survived for fourteen days, when animal inoculation, confirmatory studies on isolates from they were sacrificed. Gross pathology consisted of splenic these media included the Intensified Triple Acid-Fast Stain, enlargement, pale plaques in the liver, and nonconsolidated buffered acridine orange stain, Kinyoun's Acid-Fast Stain, nodularity in the lung. Acid-fast microcolonies were found and rhodamine-labelled muramidase.^"^ The Intensified in smears of the liver, lung (Figure 6), spleen, and blood. Acid-Fast Stain^"^° was found to be the most effective. When pooled fluid from the anterior and posterior ocular Applied to subcultures of the aqueous humor and to prepa­ chambers of two mice was examined, acid-fast micro- rations of animal tissue, ft provided sharp distinction be­ colonies were found in large numbers in both preparations tween acid-fast growth and the background. (Figures 7 and 8). Histological examination ofthe tissues is not, as yet, completed. Current work in progress in the laboratory at Wayne State University has demonstrated Results propagation in the eye of an acid-fast strain from another case of sarcoidosis. As controls, six mice received cortisone Leucocytes in the aqueous humor alone, and six were given cortisone plus uninoculated A Leishman-stained preparation of the aqueous humor medium. The control mice remained well for two weeks and showed a predominantly mononuclear infiltrate wfth ap­ no microcolonies were found in smears of tissue. proximately two leucocytes per oil immersion field. A rare neutrophil and one eosinophil were seen. This finding contrasts with the cytology in the other 63 nonsarcoid Discussion uveftis cases previously studied^"^ in which no eosinophils It is noteworthy that the aqueous humor from the anterior were seen in the aqueous humor. chamber fluid of this patient showed organisms before antibiotic therapy, whereas they were absent after antibiotic Organisms in direct smear of aqueous humor administration. Thus, therapy may have inhibited the orga­ A smearof the aqueous humor stained by Kinyoun's method nisms, either reducing them below detectable numbers or revealed a group of slender acid-fast rods (Figure 3). A altering them in a way which prevented their growth in duplicate smear stained wfth auramine-rhodamine showed culture media. colonies of fluorescent spheres. We have yet to determine whether the acid-fast organism which is associated with sarcoidosis is a new species or an Microorganisms in cultures atypical stage ofthe tubercle bacillus. The acid-fast growth Colonies did not appear on the surface of any medium. from sarcoid cases resembles tubercle bacilli in showing Growth in semi-solid agar and in pour plates was some microscopic twisted strands, the so-called pleomorphic and acid fast in the Triple Stain (Figures 1 and "cords."^'" Mankiewicz and Kurti^^ believe a phage is 2). A control for reliability ofthe Triple Stain consisted of 62 present in sarcoidosis which holds M. tuberculosis in a blood cultures containing 14 miscellaneous species of variant stage. Garvin," in the laboratory at Wayne State nonmycobacterial classical bacteria. These showed no University, has electrophoretically analyzed the proteins of acid-fast organisms. an organism in blood cultures of a sarcoidosis case. The pattern closely mimicked but did not exactly duplicate that The organisms from the patient's cultures fluoresced when of M. tuberculosis. stained wfth auramine-rhodamine, acridine orange (Figure 4), and rhodamine-labelled muramidase (Figure 5). On theother hand, the organism seen in sarcoidosis may not Acridine orange stains nucleic acids. Reaction with be M. tuberculosis. The antibodies in the serum of sar­ muramidase shows components of microbial walls, but coidosis patients react wfth varied species of Mycobacteria, does not require a complete classical wall. Most isolates not with the suggestive intensity ofM. tuberculosis.'"^ Acid- from infection of pleomorphic organisms which fail to fast species which are difficult to propagate are gaining colonize on the surface of media retain an incomplete cell increasing attention.'^'^ Also, while there is a difference wall.^ between fastidious species and cell-wall-deficient variants,

128 Barth, judge, Mattman, and Hessburg

Figs. 1 and 2 with continued incubation in Veal Infusion Agar small colonies enlarge, still retaining their acid-fast characteristic (1000X).

129 Isolation of Acid-Fast Organism from Aqueous Humor

Fig. 3 Irregular acid-fast rods in direct smear of the aqueous humor (Kinyoun's stain) (1500X).

Fig. 4 Colonies from Chanock agar cultures of the aqueous humor stained bright red with acridine orange, indicating their RNA content (1500 X).

130 Barth, Judge, Mattman, and Hessburg

Fig. 5 Microbial nature of the colonies in cultures is indicated by fluorescence of rhodamine-labelled muramidase, which has combined with mucopeptide of the cell walls (540X).

Fig. 6 Acid-fast colony from lung of mouse given subculture from uveitis case (1000X).

131 Isolation of Acid-Fast Organism from Aqueous Humor

Fig. 7 Acki-fast organisms in pooled aqueous-vitreous humors of mouse (1000X).

4

Fig. 8 Acki-fast organisms in pooled aqueous-vitreous humors of mouse inoculated with culture from patient's eye (1000X).

132 Barth, Judge, Mattman, and Hessburg

both may be difficult to propagate. Antigens of many more Wall-deficient bacteria are being isolated from a great many isolates from sarcoidosis should be analyzed by all available disease states. The animal pathogenicity of 28 microbial methods. species in the wall-deficient stage has been described.^ Wall-deficient bacteria have also been found in infected Another investigator^^ has noted auramine-rhodamine ocular sites much more frequently than in noninfected staining organisms associated with sarcoidosis, again in­ eyes.^"^ dicating the presence of Mycobacteria. Fluorescent rods were found in involved tissues of 32 patients and were absent in scalene lymph nodes of healthy persons. Conclusion Other animal models for sarcoidosis have been reported"'" In this case of uveftis, the aqueous humor contained faintly using suspensions of sarcoid tissue. Disease has been acid-fast slender rods and auramine-rhodamine staining produced, but no organisms were demonstrated by the spheres^"® suggestive of cell-wall-deficient microorganisms. methods employed. Acid-fast microcolonies in culture were inoculated into mice and retrieved from tissues of the dead or sacrificed Many questions are pertinent: Since cortisone aids remis­ animals. In staining reactions, growth characteristics and sion of both uveitis and sarcoidosis in man, how can it animal pathogenicity, the strain resembles acid-fast, cell- increase host susceptibility? It is possible that the discrep­ wall-deficient isolates from the blood of nine other sar­ ancy is related to dosage since our laboratory mice were coidosis cases. These acid-fast colon ies have not been found given maximal amounts ofthe hormone. in over 60 control blood cultures. This is the first case of Acid-fast staining of a wall-deficient mycobacterial variant sarcoidosis in which acid-fastorganisms have been found in results from two factors. First, the clinical wall-deficient the aqueous humor. They were found to colon ize in the eyes organisms retain some mural components. Second, as of inoculated mice, thus suggesting an association between Berg^° has shown, the tubercle bacillus has acid-fast the organisms and the ocular disease in sarcoidosis. cytoplasm as well as walls.

References

1. Thorn G, Adams R, Braunwald E, IsselbacherK and Petersdorf K (eds); 12. Mankiewicz E and Kurti V; Immunologic defect in patients with Harrison's Principles of Internal Medicine, ed 8. New York, McGraw- sarcoidosis, in Proceedings of Fifth International Conference on Sar­ Hill, 1977. coidosis, Levinsky L and Macholda F (eds). Prague, Universita Karlova Praha, 1971. 2. Beeson P and McDermott W (eds); Textbook of Medicine, ed 12. Philadelphia, W. B. Saunders, 1970. 13. Garvin D; Identification of cell-wall deficient forms. PhD dissertation, Wayne State University, 1975. 3. Judge M and Mattman L; Acid-fast microorganisms from the blood of sarcoidosis patients. Abstr. Annual Meeting of American Society for 14. chapman J; Mycobacterial and mycotic antibodies in sera of patients Microbiology, Atlantic City, NJ, May 4, 1976. with sarcoidosis, results of studies using agar double-diffusion tech­ nique. Ann Intern Med 55:918-924, 1961. 4. Goldman J and Girard K; Intraocular treponemes in treated congenital syphilis. Arch Ophthalmol 78:47-50, 1967. 15. Laskowski L, Marr J, Spernoga J,et al: Fastidious mycobacteria grown from porcine prosthetic-heart-valve cultures. New £ng/ j Med 5. Hessburg PC; Studies on uveitis. I. Aqueous studies. Henry Ford Hosp 297:101-102, 1977. Med I 25:255-280, 1977. 16. Manes J; Disseminated Mycobacterium kansasii infection complicat­ 6. Hessburg PC: Studies on uveitis. II. Hypotheses with case reports. ing hairy cell leukemia. yAMA 236:1878-1879,1976. Henry Ford Hosp Med / 26:17-38, 1978. 17. Richeter J, Bartak F and Halova R: Detection of mycobacteria by 7. Barth C; Studies on the etiology of uveitis; Microbiology, immunology, fluorescent microscopy in sarcoidosis, in Proceedings of Fifth Interna­ hematology. PhD dissertation, Wayne State University, 1974, pp 8-15. tional Conference on Sarcoidosis, Levinsky L and Macholda F (eds). 8. Pohlod D, Mattman L and Tunstall L; Structures suggesting cell wall Prague, Universita Karlova Praha, 1971, pp 83-84. deficient forms detected in circulating erythrocytes by fluorochrome 18. Mitchell W and Rees R; A transmissible agent from sarcoid tissue. staining. Appt Microbiol 23:225-267, 1972. Lancet 2:81, 1969.

9. Mattman L: Cell Wall Deficient Forms. Cleveland, CRC Press, 1974. 19. Taub R and Siltzback L; Induction of granulomas in mice by injection of 10. Alexander-Jackson E: A differential triple stain for demonstrating and human sarcoid and ileitis homogenates, in Proceedings of Sixth studying non-acid-fast forms ofthe tubercle bacillus in sputum, tissue, International Conference on Sarcoidosis, Iwai K and Hosoda Y (eds). New York, University Park Press, 1972, p. 20. and body fluids. Science 99:307-308, 1944.

11. Judge M: An acid-fast organism in blood or aqueous humorof Boeck's 20. BergJ; The dual nature of acid-fastness. Yale I Biol Med 26:215-223, sarcoidosis. MS thesis, Wayne State University, 1976. 1953.

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