(An)aerobic bacteria found in secondary-cataract material

Documenta Ophthalmologica 82: 125-133, 1992. �9 1992 Kluwer Academic Publishers. Printed in the Netherlands.

(An)aerobic bacteria found in secondary-cataract material A SEM/TEM study

D. K A L I C H A R A N , 1 W.L. J O N G E B L O E D , 1 L.I. LOS 2 & J.G.F. W O R S T 2

1Histology and Cell Biology Laboratory, University of Groningen; 2 Eye Clinic, University Hospital, Groningen, The Netherlands

Accepted 1 September 1992

Key words: (An)aerobic bacteria, ECCE, Golfball structure, Micro-organisms, Quiet eyes, Secondary-cataract, SEM/TEM

Abstract. Twentyfour patients, who had marked reduction of vision due to secondary-cataract developed after an ECCE, were treated by surgical cleaning of the posterior lens capsule. During this procedure globular secondary-cataract material was removed and collected for morphological examination by SEM and TEM. Fragments of various sizes and shapes, including some with a 'golfball' structure, were seen; these closely resembled particles frequently found in cataractous lenses. In addition, in 18 patients micro-organisms were found: rod-shaped bacteria, cocci, and in 2 cases yeasts. These findings were the more remarkable because these were clinically quiet eyes with no signs of intra-ocular inflammation and cultures have been persistently negative. We imagine that these bacteria must have entered the eye during the cataract extraction and have settled there without causing an infection.

Introduction

Secondary-cataract, or the renewed occurrence of opacities on the posterior lens capsule after they had appeared to be effectively removed by surgical extraction of the lens and lens cortex, is an important complication of extracapsular cataract extraction (ECCE) with or without intra-ocular lens (IOL) implantation.

Secondary-cataract formation has been described by various authors in otherwise quiet eyes [1-4]. In these eyes many different products were found in the after-cataract material, e.g., remnants of lens fibres, spherical structures, sometimes with the appearance of a golfball, erythrocytes and a mucous substance. These products are indications, in some cases, of residual vital lens material from the region of the equator and, in other cases, of residual cataractous lens material.

The equator is an area in which residual vital lens tissue can be found, in spite of good cleaning, when the posterior capsule is left in the eye.

Various cases of intra-ocular infections have been described after cataract extraction, in which clinical inflammatory reactions have always been

126

observed. The study described here concerns quiet eyes in which, in addition to secondary-cataract, micro-organisms have been found.

The purpose of our study is to describe the morphology of secondary- cataract material from the quiet eyes of 24 patients following ECCE, with the help of scanning (SEM) and transmission (TEM) electron microscopy, and to compare the results with earlier findings on secondary-cataract material obtained with SEM [1-4].

Material and methods

Clinical pre-treatment. In 24 quiet eyes of secondary-cataract patients, after the usual local anaesthesia, a limbal penetrating incision was made in the inferior temporal quadrant. A visco-elastic substance (Healon) was then injected into the anterior chamber to protect the corneal endothelium. The posterior lens capsule and the IOL were then cleaned by means of irrigation and aspiration via an irrigation/aspiration cannula which had been inserted into the posterior chamber between the posterior lens capsule and the IOL.

SEM/TEM preparation. The secondary-cataract material was then collected with the help of a glass cannula and directly placed in a 2% GA solution in 0.1 M sodium cacodylate buffer (pH 7.4; 4~ After prefixation with GA the secondary-cataract material was transferred to a 0.22 p~m Millipore filter and rinsed three times with sodium cacodylate+ 6.8% sucrose, after which it was postfixed with 1% OsO 4 in cacodylate buffer (pH 7.4; 4~ 1 hr). After rinsing with this buffer solution, double distilled water and ethanol successively, the secondary-cataract material was dried in air. One half of the filter was made conductive for SEM examination [JEOL SEM, 15- 25 kV] by sputtercoating with an Au layer (15 nm). The other half of the filter was impregnated via a vacuum (100mmHg) with Epon 812, after which ultra-thin sections were cut and treated with uranyl acetate/lead citrate for TEM examination [Akashi 002A, 80 kV].

Results

Figure i gives a total overview of the secondary-cataract material on the filter, showing large angular pieces, more rounded portions, mucus-like material containing erythrocytes, pieces of inflammatory material and entities with a 'golfball' structure.

An unexpected finding was the presence of various micro-organisms, which at this magnification, however, were not clearly visible. One type of micro-organism, viz. the yeasts, can be seen in various stages of cell-division in Fig. 2, in addition rod-shaped bacteria, patches of mucus with cocci in and on them, and here and there erythrocytes are spread over the filter

127

Plate I.

Fig. 1. Overview of secondary-cataract material collected on 0.24 ~m Millipore filter, with mucous residue (sl), fragments (*) of various sizes, some with golfball structure (gb), erythrocytes (er). Magn. • Fig. 2. Portion of filter (fi) with yeasts (ye) in various stages of cell division, cocci (co), rod-shaped bacteria (rb), partially embedded in mucus. Magn. • Fig. 3. Higher magnification of colony of cocci (co). Note relatively rough surface structure and attachment of cocci by mucus (threads) (sl). Magn. • Fig. 4. T]EM image of section of round bacterium with relatively thick layered wall (la) and several vacuoles (va) in the cytoplasm (cy). Magn. x34,000.

128

Plate I1. Fig. 5. Magnification of SEM image of golfball structure (*) with rod-shaped bacteria. Magn. x 11,000. Fig. 6. Detail of golfball structure, surface shows holes (*) of more or less uniform size, in some of which micro-organisms (mi) are visible. Magn. x4500. Fig. Z Detail of golfball structure with partly perforated (po), partly smooth surface. Magn. x4500. Fig. 8. TEM section of golfball structure with very little contents, surrounded by a thin, slightly wavy membrane (me). Magn. x13,500.

129

surface. In Fig. 3 a cluster of cocci of various sizes, with a rough surface structure, are visible; the cocci appear to be stuck together with mucus. The section examined with TEM, Fig. 4, makes it clear that these structures are bacteria. A firm wall surrounds the cytoplasm, which shows strong contrast thanks to absorption of osmium. The wall appears to consist of layers and the cytoplasm shows vacuoles, the contents of some of which seem to have been extracted.

Figure 5 shows a detail of the golfball structure seen in Fig. 1: the indentations/flattened facets vary in size and cocci or rod-shaped bacteria are sometimes attached to them. The erythrocytes which are also present can easily be distinguished by their size and biconcave shape. Figure 6 shows a higher magnification of the golfball structure with a surface which varies 'in its degree of roughness; micro-organisms are visible in the holes and appear to be attached with mucus to the less rough surfaces. Figure 7 shows a detail of a smaller golfball structure, the surface seems to be somewhat irregular and porous in character. Figure 8 is the corresponding TEM picture; it is clear that large irregular vesicles can be seen here, which have very little contents but are surrounded by a membrane. They are arranged neatly on the filter surface.

Figure 9 shows rough-looking rod-shaped bacteria spread out over an erythrocyte. Note the thin filamentous projections of mucous/protein material which, by cross-linking with the GA fixative, are attached to the surface of the erythrocytes.

Figure 10, a TEM image, shows two rod-shaped bacteria cut open in the long axis. The thin electron-dense cell membrane encloses the cytoplasm with its structures, such as vacuoles partially filled with a nuclear network.

In Fig. 11, a SEM image, other kinds of rod-shaped bacteria are seen. They exhibit more pleomorphism and irregularities than those seen in Fig. 9, while their surfaces are smoother and not covered with mucus. The capsular sheath clearly exhibits various sorts of protuberances.

Figure 12 shows a number of clearly defined, somewhat oval structures in the secondary-cataract material against the background (substrate) of porous Millipore filter material. These structures are very uniform in size and brightness, as represented by SEM.

Discussion

From the observations described here it is clear that more is going on in quiet eyes after an extracapsular cataract extraction than would appear at first sight. The patients examined did not have inflamed eyes, only visual problems on account of secondary-cataract. A large proportion of operated cataract patients (J.G.F. Worst, personal communication) suffer from reduced vision in the course of time on account of secondary-cataract [5]. Globular secondary-cataract is treated by Worst by lavage of the posterior

130

Plate III. Fig. 9. Rod-shaped bacteria (rb) with rough surfaces and threadlike projections (arrows) on the surface of an erythrocyte (er). Magn. • Fig. 10. TEM section of rod-shaped bacterium cut open in the long axis, with vacuoles (va) and nuclear filaments (nf) in the cytoplasm. Magn. • Fig. 11. Smooth rod-shaped bacteria (rb) with bulging (arrows) cytoplasm. Magn. • Fig. I2. Overview of secondary-cataract material with typical oval structures (ov) with strong contrast, measuring -+ I ~m. Magn. • 11,000.

131

lens capsule. During the operation garamycin is injected subconjunctivally. In order to make the risk of contamination as slight as possible an airflow system is used. The skin and lashes of the patient are completely covered with Steridrape in such a way that no pockets of fluid can form between the Steridrape and the skin. As no gloves are worn a non-touch technique is used (tips of instruments and needles are not touched by the fingers). These preventive measures make infection by this route almost impossible [6]. In addition, during the operation filtered fluids only are used.

It is still not clear how the micro-organisms can have entered the eye during the operation in spite of the sterile conditions. One possibility is that the micro-organisms derive from the fornix conjunctivae of the patient himself and enter the eye during the operation. Thus the micro-organisa- tions found in secondary-cataract material may have already become encapsulated in remnants of residual cataractous lens material.

In an earlier study [4] it was seen that similar golfball structures were present in cataract lenses and that they were degradation products of cataractous lens fibres. These particles seem to have been left behind after the lens extraction, without at first causing problems for the patient. Cusumano et al. [7] also found these particles and called them starch-like granules with bacteria attached to their surface. Our findings are that they are no degeneration products of degenerating lens fibres. In view of the poor contrast with SEM, the particles appear not to absorb much osmium, so it is likely that they are composed of starch or protein-like material. That the material is soft and easy to dent appears from the pictures, in which impressions of other, smaller spherical structures can be clearly seen. The more rounded or somewhat oval structures, on the other hand, absorb much osmium, especially when a non-coating technique, like the TAO method, is used; presumably they have lipid contents.

The above considerations relate to clinically quiet eyes. Clinically mani- fest intra-ocular inflammation can be subdivided into 3 categories: acute, chronic and low grade.

The acute form usually occurs directly after the operation and can be cured within a few weeks by treatment with medicines such as corticos- teroids and antibiotics.

The chronic form, or toxic lens syndrome, is probably caused by toxins which are released after the implantation of biomaterial, such as an IOL or sutures made of synthetic material, e.g., nylon, supramid, etc. [1-3, 8-12]. Notwithstanding the sterile conditions during implantation, the released toxic components cause inflammation in the long run, so that these synthetic materials are in fact not, or not completely, biocompatible.

The so-called low grade inflammation has been described by various authors [13-18]. There are three main causes of low grade inflammation: (a) the patient's own flora [19, 20]; (b) surgical irrigating and IOL neutralizing fluids [21-23] and (c) contamination of surgical instruments and other aids, which may also give rise to epidemic ocular infections [24, 25].

132

The source of the bacteria found by us is presumably the conjunctiva, where Propionibacteria are present; on the other hand, Staphylococcus epidermidis is to be found on the skin. The cultures were negative, but it is not certain whether the culture medium and/or the incubation time were ideal.

A possible adaptation of the technique would be to rinse the fornix conjunctivae with balanced saline and, if necessary, local application of iodine.

References

1. Jongebloed WL, Dijk F, Kruis J, Worst JGF. Soemmering's ring, an aspect of secondary cataract: A morphological description by SEM. Doc Ophthalmol 1988; 70: 165-70.

2. Jongebloed WL, van der Veen G, Dijk F, Worst JGF. Secondary cataract material collected with a glass cannula: a SEM study. Doc Ophthalmol 1990; 75: 359-64.

3. Jongebloed WL, van der Veen G, Kalicharan D, Rijneveld WJ, Houtman WA, Worst JGF. Reaction of the rabbit corneal endothelium to nylon sutures: A SEM study. Doc Ophthalmol 1990; 75: 351-58.

4. Jongebloed WL, Kalicharan D, Los LI, van der Veen G, Worst JGF. A combined scanning and transmission electronmicroscopic investigation of human (secondary) cataract material. Doc Ophthalmol 1991; 78: 325-34.

5. Sawusch MR, McDonnell P. Posterior capsule opacification. Current Opinion in Ophthal- mology 1990; 1: 28-33.

6. Worst JGF, Los LI. Some aspects of implant surgery. Eur J Implant Ref Surg 1991; 3: 157-67.

7. Cusumano A, Massimo B, Spitznas M. Is chronic intraocular inflammation after lens implantation of bacterial origin? Ophthalmol 1991; 11: 1703-10.

8. Apple DJ, Mamalis N, Lottfield K, et al. Complications of intraocular lenses: A histological and histopathological review. Surv Ophthalmol 1984; 29: 1-54.

9. Hunter JW. Early postaphative sterile hypopyons. Br J Ophthalmol 1978; 62: 470-73. 10. Jongebloed WL, Figueras MJ, Humalda D, Blanksma LJ, Worst JGF. Mechanical and

biochemical effects of man-made fibres and metals in the human eye: A SEM study. Doc Ophtbalmol 1986; 61: 304-12.

11. Galin MA, Goldstein IM, Tuberville A, et al. Intraocular lenses generate chemotactic activity in human serum. Binkhorst Lecture Part II. Arch Ophthalmol 1981; 99: 1434-35.

12. Kenneth LP, Apple DJ, Kincaid MC, et al. Localized endophthalmitis: A newly described cause of so-called toxic lens syndrome. J Cataract Refract Surg 1987; 13: 498-510.

13. Neumann AC. Particulate and microbial contamination of intraocular irrigation solutions. J Cataract Refract Surg 1986; 12: 485-88.

14. Wirostko E, Johnson L, Wirostko B. Postinflammatory cataracts in the mouse: Induction by human mycoplasma-like organisms. Br J Ophthalmol 1991; 75: 671-74.

15. Meisler DM, Palestine AG, Vastine DW, et al. Chronic propionibacterium endophthalmitis after extracapsular cataract extraction and intraocular lens implantation. Am J Ophthalmol 1986; 102: 733-39.

16. Pettit TH, Olson RJ, Foos RY, et al. Fungal endophthalmitis following intraocular lens implantation; surgical epidemic. Arch Ophthalmol 1980; 98: 1025-39.

17. Stern WH, Tamura E, Jacobs RA, et al. Epidemic postsurgical candida parapsilosis endophthalmitis: Clinical findings and management of 15 consecutive cases. Ophthalmology 1985; 92: 1701-9.

133

18. Driebe WT, Mandelbaum S, Forster RK, et al. Pseudophakic endophthalmitis: Diagnosis and management. Ophthalmology 1986; 93: 442-498.

19. Walker CB, Claou6 Cm. Incidence of conjunctional colonization by bacteria capable of causing postoperative endophthalmitis. J Soc Med 1986; 79: 520-21.

20. Jaffe GJ, Whitcher JP, Biswell R, et al. Propionibacterium acnes endophthahnitis seven months after extracapsular cataract extraction and intraocular lens implementation. Ophthalmic Surg 1986; 17: 791-93.

21. Samples JR, Buider PS. Contamination of migrating solution used for cataract surgery. Ophthalmic Surg 1984; 15: 66.

22. O'Day DM. Value of a centralized surveillance system during a national epidemic of endophthalmitis. Ophthalmology I985; 92: 309-15.

23. Neumann AC, Dzelzkalns JJ, Bessinger DJ. J Cataract Refract Surg 1991; 17: 353-58. 24. Meltner DW. Sterile hypopyon following intraocular lens surgery. Arch Ophthalmol 1980;

98: 100-104. 25. Richburg FA, Reidy JJ, Apple DJ, et al. Sterile hypopyon secondary to ultrasonic cleaning

solution. J Cataract Refrac Surg 1986; 12: 248-51.

Address for correspondence: Dr D. Kalicharan, Histology and Cell Biology Laboratory, University of Groningen, Oostersingel 69/1, 9713 EZ Groningen, The Netherlands. Phone: (50) 632 501 / 632 495 / 632 499.