Isolation of an Acid-Fast Organism from the Aqueous Humor

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.. Hessburg, MD * * * The anterior chamber fluid from

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

cell-wall-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

uveitis-sarcoidosis 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 aque-ous humor appears to be sterile by routine culture methods Similarly, in the case described here no classical microorga-nisms appeared in a battery of media for aerobes, an-aerobes 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 im-proved staining and culture methods diagnosis of sar-coidosis-and its complications may be facilitated

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

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 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

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

simul-taneously given cortisone subcusimul-taneously 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

prepa-rations of animal tissue, ft provided sharp distinction

be-tween acid-fast growth and the background

Results Leucocytes in the aqueous humor

A Leishman-stained preparation of the aqueous humor

showed a predominantly mononuclear infiltrate wfth

ap-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

uveftis cases previously studied^"^ in which no eosinophils

were seen in the aqueous humor

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

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 p o u r plates was

pleomorphic and acid fast in the Triple Stain (Figures 1 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)

A c r i d i n e orange stains n u c l e i c 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.^

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

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 micro-colonies were found in large numbers in both preparations (Figures 7 and 8) Histological examination o f t h e 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

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 orga-nisms, either reducing them below detectable numbers or altering them in a way which prevented their growth in culture media

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

O n theother hand, the organism seen in sarcoidosis may not

be M tuberculosis The antibodies in the serum of sar-coidosis patients react wfth varied species of Mycobacteria, not with the suggestive intensity o f M tuberculosis.'"^ Acid-fast species which are difficult to propagate are gaining increasing attention.'^'^ Also, while there is a difference between fastidious species and cell-wall-deficient variants,

Barth, judge, Mattman, and Hessburg

Figs 1 and 2

w i t h continued incubation in Veal Infusion Agar small colonies enlarge, still retaining their acid-fast characteristic

(1000X)

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)

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)

Isolation of Acid-Fast Organism f r o m 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)

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

Another investigator^^ has noted auramine-rhodamine

staining organisms associated with sarcoidosis, again

in-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

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

Many questions are pertinent: Since cortisone aids

remis-sion of both uveitis and sarcoidosis in man, how can it

increase host susceptibility? It is possible that the

discrep-ancy is related to dosage since our laboratory mice were

given maximal amounts o f t h e 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 s h o w n , the t u b e r c l e b a c i l l u s has acid-fast

cytoplasm as well as walls

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.^"^

Conclusion

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, cell-wall-deficient isolates from the blood of nine other sar-coidosis 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

References

1 Thorn G, Adams R, Braunwald E, IsselbacherK and Petersdorf K (eds);

Harrison's Principles of Internal Medicine, ed 8 New York,

McGraw-Hill, 1977

2 Beeson P and McDermott W (eds); Textbook o f Medicine, ed 12

Philadelphia, W B Saunders, 1970

3 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

4 Goldman J and Girard K; Intraocular treponemes in treated congenital

syphilis Arch Ophthalmol 78:47-50, 1967

5 Hessburg PC; Studies on uveitis I Aqueous studies Henry Ford Hosp

Med I 25:255-280, 1977

6 Hessburg PC: Studies on uveitis II Hypotheses w i t h case reports

Henry Ford Hosp Med / 26:17-38, 1978

7 Barth C; Studies on the etiology of uveitis; Microbiology, immunology,

hematology PhD dissertation, Wayne State University, 1974, pp 8-15

8 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

9 Mattman L: Cell Wall Deficient Forms Cleveland, CRC Press, 1974

10 Alexander-Jackson E: A differential triple stain for demonstrating and

studying non-acid-fast forms o f t h e tubercle bacillus in sputum, tissue,

and body fluids Science 99:307-308, 1944

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

12 Mankiewicz E and Kurti V; Immunologic defect in patients w i t h sarcoidosis, in Proceedings of Fifth International Conference on Sar-coidosis, 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

w i t h sarcoidosis, results of studies using agar double-diffusion tech-nique Ann Intern Med 55:918-924, 1961

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 complicat-ing hairy cell leukemia yAMA 236:1878-1879,1976

17 Richeter J, Bartak F and Halova R: Detection of mycobacteria by fluorescent microscopy in sarcoidosis, in Proceedings of Fifth Interna-tional Conference on Sarcoidosis, Levinsky L and Macholda F (eds) Prague, Universita Karlova Praha, 1971, pp 83-84

18 Mitchell W and Rees R; A transmissible agent from sarcoid tissue Lancet 2:81, 1969

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

20 BergJ; The dual nature of acid-fastness Yale I Biol M e d 26:215-223,

1953