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 sarcoidosis was found to contain microcolonies of a cellwall-deficient organism that was propagated and identified as acid-fast by the Intensified Acid-Fast stain The colonies were inoculated into mice and retrieved from the dead or sacrificed animals This report suggests that the acid-fast microbe in the aqueous humor of this case of uveitissarcoidosis may be the same organism as that found in the blood in sarcoidosis Thus, it may be associated not only with the primary disease, but also with the complications of Boeck's sarcoidosis Introduction A n t e r i o r uveitis is frequently a local manifestation of systemic Boeck's sarcoidosis.^-^ In fact, an early name for sarcoidosis was uveoparotid fever In such cases the aqueous humor appears to be sterile by routine culture methods Similarly, in the case described here no classical microorganisms appeared in a battery of media for aerobes, anaerobes Mycoplasma, Mycobacteria, and fungi However, 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 improved staining and culture methods diagnosis of sarcoidosis-and its complications may be facilitated Case Report Submitted for publication; October 2, 1978 Accepted for publication; February 19,1979 * Department of Pathology, Henry Ford Hospital ** Department of Biology, Wayne State University, Detroit, Michigan *** Detroit Institute of Ophthalmology, Grosse Pointe Park, Michigan Address reprint requests to Dr Barth, Departmentof Pathology, Henry Ford Hospital, 2799 W Grand Blvd, Detroit, Ml 48202 127 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 weeks and then discontinued The eye remained quiet for ten months after antibiotics were stopped At that time the anterior uveitis recurred and a second paracentesis was performed on the left eye Cell-wall-deficient forms were isolated only from cultures of the anterior chamber fluid taken during the flrst paracentesis This report concerns on ly those organisms propagated from the flrst sample of aqueous humor Isolation of Acid-fast Organism from Aqueous Humor Special Laboratory Studies Growth occurred only at 37°C rather than at 25°C and was minimal under anaerobic conditions Hypertonic medium containing 10% sucrose did not improve growth Microdrops (0.03 ml) of anterior chamber fluid were treated as described in a previousreport.^'^AIiquotsof growth inthe Medill-O'Kane Broth were frozen and later subcultured to Kirschner's broth and Horse Muscle broth^ for animal inoculation Six mice were inoculated intraperitoneally with cuftures of the acid-fast organism and were simultaneously given cortisone subcutaneously In addftion to animal inoculation, confirmatory studies on isolates from these media included the Intensified Triple Acid-Fast Stain, buffered acridine orange stain, Kinyoun's Acid-Fast Stain, and rhodamine-labelled muramidase.^"^ The Intensified Acid-Fast Stain^"^° was found to be the most effective Applied to subcultures of the aqueous humor and to preparations of animal tissue, ft provided sharp distinction between acid-fast growth and the background Animal inoculation Four mice given the acid-fast culture and a cortisone dosage of 50 mg died between eight and eleven days Two mice given 40 mg of cortisone survived for fourteen days, when they were sacrificed Gross pathology consisted of splenic enlargement, pale plaques in the liver, and nonconsolidated nodularity in the lung Acid-fast microcolonies were found in smears of the liver, lung (Figure 6), spleen, and blood When pooled fluid from the anterior and posterior ocular chambers of two mice was examined, acid-fast microcolonies were found in large numbers in both preparations (Figures and 8) Histological examination ofthe tissues is not, as yet, completed Current work in progress in the laboratory at Wayne State University has demonstrated propagation in the eye of an acid-fast strain from another case of sarcoidosis As controls, six mice received cortisone alone, and six were given cortisone plus uninoculated medium The control mice remained well for two weeks and no microcolonies were found in smears of tissue Results Leucocytes in the aqueous humor A Leishman-stained preparation of the aqueous humor showed a predominantly mononuclear infiltrate wfth approximately 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 uveftis cases previously studied^"^ in which no eosinophils were seen in the aqueous humor Discussion It is noteworthy that the aqueous humor from the anterior chamber fluid of this patient showed organisms before antibiotic therapy, whereas they were absent after antibiotic administration Thus, therapy may have inhibited the organisms, either reducing them below detectable numbers or altering them in a way which prevented their growth in culture media Organisms in direct smear of aqueous humor A smearof the aqueous humor stained by Kinyoun's method revealed a group of slender acid-fast rods (Figure 3) A duplicate smear stained wfth auramine-rhodamine showed 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 atypical stage ofthe tubercle bacillus The acid-fast growth from sarcoid cases resembles tubercle bacilli in showing some microscopic twisted strands, the so-called "cords."^'" Mankiewicz and Kurti^^ believe a phage is present in sarcoidosis which holds M tuberculosis in a variant stage Garvin," in the laboratory at Wayne State University, has electrophoretically analyzed the proteins of an organism in blood cultures of a sarcoidosis case The pattern closely mimicked but did not exactly duplicate that of M tuberculosis Microorganisms in cultures Colonies did not appear on the surface of any medium G r o w t h in semi-solid agar and in pour plates was pleomorphic and acid fast in the Triple Stain (Figures and 2) A control for reliability ofthe Triple Stain consisted of 62 blood cultures containing 14 miscellaneous species of nonmycobacterial classical bacteria These showed no acid-fast organisms The organisms from the patient's cultures fluoresced when stained wfth auramine-rhodamine, acridine orange (Figure 4), and r h o d a m i n e - l a b e l l e d muramidase (Figure 5) Acridine orange stains nucleic acids Reaction w i t h muramidase shows components of microbial walls, but does not require a complete classical wall Most isolates from infection of pleomorphic organisms which fail to colonize on the surface of media retain an incomplete cell wall.^ On theother hand, the organism seen in sarcoidosis may not be M tuberculosis The antibodies in the serum of sarcoidosis patients react wfth varied species of Mycobacteria, not with the suggestive intensity o f M tuberculosis.'"^ Acidfast species which are difficult to propagate are gaining increasing attention.'^'^ Also, while there is a difference between fastidious species and cell-wall-deficient variants, 128 Barth, judge, Mattman, and Hessburg Figs and w i t h 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 Irregular acid-fast rods in direct smear of the aqueous humor (Kinyoun's stain) (1500X) Fig 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 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 Acid-fast colony from lung of mouse given subculture from uveitis case (1000X) 131 Isolation of Acid-Fast Organism from Aqueous Humor Fig Acki-fast organisms in pooled aqueous-vitreous humors of mouse (1000X) Fig 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 isolates from sarcoidosis should be analyzed by all available methods Wall-deficient bacteria are being isolated from a great many disease states The animal pathogenicity of 28 microbial species in the wall-deficient stage has been described.^ Wall-deficient bacteria have also been found in infected ocular sites much more frequently than in noninfected eyes.^"^ Another investigator^^ has noted auramine-rhodamine staining organisms associated with sarcoidosis, again indicating 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 r e p o r t e d " ' " using suspensions of sarcoid tissue Disease has been produced, but no organisms were demonstrated by the methods employed In this case of uveftis, the aqueous humor contained faintly acid-fast slender rods and auramine-rhodamine staining spheres^"® suggestive of cell-wall-deficient microorganisms Acid-fast microcolonies in culture were inoculated into mice and retrieved from tissues of the dead or sacrificed animals In staining reactions, growth characteristics and animal pathogenicity, the strain resembles acid-fast, cellwall-deficient isolates from the blood of nine other sarcoidosis cases These acid-fast colon ies have not been found in over 60 control blood cultures This is the first case of sarcoidosis in which acid-fastorganisms have been found in the aqueous humor They were found to colon ize in the eyes of inoculated mice, thus suggesting an association between the organisms and the ocular disease in sarcoidosis Many questions are pertinent: Since cortisone aids remission of both uveitis and sarcoidosis in man, how can it increase host susceptibility? It is possible that the discrepancy is related to dosage since our laboratory mice were given maximal amounts ofthe hormone Acid-fast staining of a wall-deficient mycobacterial variant results from two factors First, the clinical wall-deficient organisms retain some mural components Second, as Berg^° has shown, the tubercle bacillus has acid-fast cytoplasm as well as walls References Thorn G, Adams R, Braunwald E, IsselbacherK and Petersdorf K (eds); Harrison's Principles of Internal Medicine, ed New York, McGrawHill, 1977 Beeson P and McDermott W (eds); Textbook of Medicine, ed 12 Philadelphia, W B Saunders, 1970 Judge M and Mattman L; Acid-fast microorganisms from the blood of sarcoidosis patients Abstr Annual Meeting of American Society for Microbiology, Atlantic City, NJ, May 4, 1976 12 Mankiewicz E and Kurti V; Immunologic defect in patients with sarcoidosis, in Proceedings of Fifth International Conference on Sarcoidosis, Levinsky L and Macholda F (eds) Prague, Universita Karlova Praha, 1971 13 Garvin D; Identification of cell-wall deficient forms PhD dissertation, Wayne State University, 1975 14 chapman J; Mycobacterial and mycotic antibodies in sera of patients with sarcoidosis, results of studies using agar double-diffusion technique Ann Intern Med 55:918-924, 1961 Goldman J and Girard K; Intraocular treponemes in treated congenital syphilis Arch Ophthalmol 78:47-50, 1967 Hessburg PC; Studies on uveitis I Aqueous studies Henry Ford Hosp Med I 25:255-280, 1977 15 Laskowski L, Marr J, Spernoga J,et a l : Fastidious mycobacteria grown f r o m p o r c i n e p r o s t h e t i c - h e a r t - v a l v e c u l t u r e s N e w £ng/ j M e d 297:101-102, 1977 16 Manes J; Disseminated Mycobacterium kansasii infection complicating hairy cell leukemia yAMA 236:1878-1879,1976 Hessburg PC: Studies on uveitis II Hypotheses with case reports Henry Ford Hosp Med / 26:17-38, 1978 17 Richeter J, Bartak F and Halova R: Detection of mycobacteria by fluorescent microscopy in sarcoidosis, in Proceedings of Fifth International Conference on Sarcoidosis, Levinsky L and Macholda F (eds) Prague, Universita Karlova Praha, 1971, pp 83-84 Barth C; Studies on the etiology of uveitis; Microbiology, immunology, hematology PhD dissertation, Wayne State University, 1974, pp 8-15 Pohlod D, Mattman L and Tunstall L; Structures suggesting cell wall deficient forms detected in circulating erythrocytes by fluorochrome staining Appt Microbiol 23:225-267, 1972 18 Mitchell W and Rees R; A transmissible agent from sarcoid tissue Lancet 2:81, 1969 Mattman L: Cell Wall Deficient Forms Cleveland, CRC Press, 1974 19 Taub R and Siltzback L; Induction of granulomas in mice by injection of human sarcoid and ileitis homogenates, in Proceedings of Sixth International Conference on Sarcoidosis, Iwai K and Hosoda Y (eds) New York, University Park Press, 1972, p 20 10 Alexander-Jackson E: A differential triple stain for demonstrating and studying non-acid-fast forms ofthe tubercle bacillus in sputum, tissue, and body fluids Science 99:307-308, 1944 20 BergJ; The dual nature of acid-fastness Yale I Biol Med 26:215-223, 1953 11 Judge M : An acid-fast organism in blood or aqueous h u m o r o f Boeck's sarcoidosis MS thesis, Wayne State University, 1976 133 ... Isolation of Acid-Fast Organism from Aqueous Humor Fig Irregular acid-fast rods in direct smear of the aqueous humor (Kinyoun's stain) (1500X) Fig Colonies from Chanock agar cultures of the aqueous. .. Isolation of Acid-Fast Organism from Aqueous Humor Fig Acki-fast organisms in pooled aqueous- vitreous humors of mouse (1000X) Fig Acki-fast organisms in pooled aqueous- vitreous humors of mouse... were seen in the aqueous humor Discussion It is noteworthy that the aqueous humor from the anterior chamber fluid of this patient showed organisms before antibiotic therapy, whereas they were absent