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Propionibacterium acnes DNA from an explanted intraocular lens detected by polymerase chain reaction in a case of chronic pseudophakic endophthalmitis
J CATARACT REFRACT SURG - VOL 32, MARCH 2006
Propionibacterium acnes DNA from an explanted intraocular lens detected by polymerase chain reaction in a case of chronic pseudophakic endophthalmitis Jui-Yang Lai, MS, Ko-Hua Chen, MD, Ying-Cheng Lin, MD, Wen-Ming Hsu, MD, Shui-Mei Lee, MD
We report a case of Propionibacterium acnes DNA from the explanted intraocular lens (IOL) a 76-year-old man with delayed-onset chronic endophthalmitis. The detection was by polymerase chain reaction (PCR). Slitlamp microscopic examination showed cells and flare in the anterior chamber, keratic precipitates on the endothelium, and multiple white plaques on both surfaces of the IOL. Propionibacterium acnes DNA was found in both the aqueous humor and the explanted IOL by PCR. Scanning electron microscopy showed diphtheroid particles, a coating of biofilm-like deposits, as well as scattered crystals on the surface of the explanted IOL and supported the possibility of P acne colonies on the IOL. J Cataract Refract Surg 2006; 32:522–525 Q 2006 ASCRS and ESCRS
Propionibacterium acnes infection is a main cause of delayed-onset chronic endophthalmitis associated with cataract surgery.1 The exact mechanisms by which the bacterium P acnes causes endophthalmitis after cataract surgery are not completely known. The binding of bacteria on the surface of the intraocular lens (IOL) during implantation, with its concealment between the IOL optic and capsular bags, is known to play a role in the pathogenesis of pseudophakic endophthalmitis.2 Evidence supporting this hypothesis comes from scanning electron microscopy
Accepted for publication July 11, 2005. From the Department of Chemical Engineering (Lai), National Tsing Hua University, Hsinchu, and the Division of Medical Engineering (Lai, Chen), National Health Research Institutes, Department of Ophthalmology (Chen, Lin, Hsu, Lee), Taipei Veterans General Hospital, National Yang Ming University (Chen, Hsu), Taipei, Taiwan, China. Supported by VGH-93-360-5 from the Taipei Veterans General Hospital. No author has a financial or proprietary interest in any material or method mentioned. Reprints requests to Ko-Hua Chen, MD, Department of Ophthalmology, Taipei Veterans General Hospital, 201 Shih-Pai Road, Section 2, Taipei, Taiwan 11217, China. E-mail: khchen@vghtpe. gov.tw. Q 2006 ASCRS and ESCRS Published by Elsevier Inc.
522
(SEM) morphologic studies.3 Positive cultures of P acnes came from the excised posterior capsular bag and explanted IOL,2,4 although the culture rate of P acnes was low.5 Furthermore, misinterpretation of IOL observations by SEM is possible.6 Polymerase chain reaction (PCR) has been shown to be a more sensitive method than culturing to detect intraocular P acnes infection in aqueous and vitreous samples from cases of delayed-onset endophthalmitis.7,8 However, PCR has not been used to detect P acnes on IOLs. Scanning electron microscopy has been used to demonstrate bacterial adherence to the IOL in studies of pseudophakic endophthalmitis.3,6 We report a case of delayed-onset chronic endophthalmitis and P acnes DNA from an explanted IOL detected by PCR. The images of the IOL surfaces on SEM examination support the possibility of bacterial adherence on the IOL. CASE REPORT A 76-year-old man presented to our department with progressive blurred vision, redness, tearing, and photophobia in the left eye. His history was remarkable for delayed-onset chronic endophthalmitis and secondary glaucoma after cataract surgery in the left eye since May 2001. In January 2001, he had cataract phacoemulsification surgery in the left eye, during which posterior capsule rupture occurred. An anterior chamber IOL (1-piece poly(methyl methacrylate) 4-point fixation, Alcon) was implanted. Before the 0886-3350/06/$-see front matter doi:10.1016/j.jcrs.2005.12.071
CASE REPORTS: PROPIONIBACTERIUM ACNES ON EXPLANTED IOL
surgery, cephalexin (250 mg every 6 hours) was prescribed for prophylaxis of infection. No antibiotics were added to the irrigation solution. Fifteen milligrams gentamicin subconjunctival injection was given postoperatively. Two months later, the left eye developed redness, pain, and blurred vision. Delayed-onset endophthalmitis was diagnosed by his surgeon, and in Febuary 2002, he was referred to our hospital for further management. On examination, the best corrected visual acuity (BCVA) in the left eye was 20/1200. Slitlamp microscopic examination demonstrated congestion of the left conjunctiva, mild stromal edema of the left cornea, cells and flare in the aqueous humor, keratic precipitates on the corneal endothelial layer, and white plaquelike deposits on both surfaces of the anterior chamber of the IOL (Figure 1). The posterior capsule was absent. After unsuccessful treatment with topical and systemic vancomycin and corticosteroids for 2 weeks, the patient had another surgery to remove the IOL in the anterior chamber and vitrectomy performed at our hospital. Before the surgery, the cultures from the conjunctiva and aqueous humor sample were taken. The 0.1 mL aqueous humor sample was sent for PCR detection of P acnes. An aqueous humor sample from a healthy cataract patient and a new IOL were used as the negative controls. After surgery, all signs and symptoms of endophthalmitis subsided. The BCVA improved to 20/40 with spectacles at 2 months follow-up. The explanted IOL was bisected and immediately submitted to the pathology laboratory in a dry, sterile container. Half the sample was sent for PCR, and the other half was immersed in Karnovsky fixation solution and processed for SEM (Model JSM 5600; Jeol). For SEM examination, the IOL optics were cut with a blade into 5, 2.0 mm31.5 mm31.0 mm3 pieces, which then were subjected to a series of fixation and dehydration treatments. Each piece was subsequently mounted onto an aluminum stub and coated with gold in a sputter coater. One piece was analyzed with an energy dispersive x-ray spectroscopy (Oxford Instruments America, Inc.) to define the nature of the crystals on the IOL.
Polymerase chain reaction was performed to amplify 16S rRNA gene fragments of P acnes. Antisense primer (50 -GGGTTGATAA(A/T)CCGCTTTCGCCTG-30 ) and sense primer (50 -GGGACACCCATCTCTGAGCAC-30 ) were designed specifically to amplify a 587 bp portion of the P acnes 16S rRNA gene (GenBank accession number PA16SLIP4).9 Using 5 mL extracted DNA samples, the following conditions were used for amplification: initial denaturation 94 C for 4 minutes and then 94 C for 30 seconds, 50 C for 30 seconds, and 72 C for 1 minute for 45 cycles. Alhough all cultures from the conjunctival swab, vitreous, and aqueous humor failed to demonstrate P acnes, P acnes DNA was detected from both the aqueous humor sample and the explanted IOL (Figure 2). Under SEM, a biofilm-like coating and scattered crystals were found spread over both sides of the optic surface from the explanted IOL (Figure 3, A). Diphtheroid particles with sizes equal to or smaller than approximately 0.6 mm and their morphologic characteristics were compatible with those in previous reports (Figure 3, B). It was noted that a few crystallike deposits were scattered on both surfaces of the IOL optics (Figure 3, C). From energy dispersive x-ray spectroscopic analyses, the crystal-like deposits were shown to be composed of sodium and chloride (Figure 3, D). DISCUSSION
Clinically, P acnes endophthalmitis manifested as a chronic iridocyclitis is characterized by granulomatousappearing keratic precipitates, hypopyon, and white plaques on the posterior capsule or IOL.10,11 The diagnosis of P acnes infection in pseudophakic endophthalmitis can be difficult because the culture results from the aqueous humor are usually negative due to the slow growth rate of M
1
2
3
4
5
500 bp-
Figure 1. Two weeks after treatment with systemic and topical vancomycin and corticosteroids, white plaque deposits on the IOL, cells and flare in the anterior chamber, and keratic precipitates on the endothelium were seen on by slitlamp microscopy.
Figure 2. Electrophoresis of PCR amplification products using primers for 1 587 bp portion of the P acnes 16S rRNA gene. Lane 1: 5 mL DNA extracted from the explanted IOL. Lane 2: Aqueous samples from the patient. Lane 3: Positive control. Lane 4: Aqueous humor from a healthy cataract eye as a negative control. Lane 5: A new IOL as a negative control. Marker (M) is a 100 bp ladder.
J CATARACT REFRACT SURG - VOL 32, MARCH 2006
523
CASE REPORTS: PROPIONIBACTERIUM ACNES ON EXPLANTED IOL
Figure 3. Scanning electron microscopic photographs showed the 3-dimensional view of the lens surface. A granule-like coating on the surface of the IOL was noted under lower manifestation (A). Upon higher manifestation, diphtheroid particles of !1 mm size were spread evenly over the whole surface (B), and scattered crystals were also noted (C). A: Original magnification, 500. B and C: Original magnification 2000. Energy dispersive x-ray spectroscopic analyses of the explanted IOL from this patient showed that sodium and chloride were distributed on the IOL surface (D). Note only carbon and oxygen on the optics and haptics of the control (data not shown).
P acnes5 and also possibly due to the sequestered state of the organisms in the remaining capsular bag.2 To increase the detection rate of P acnes responsible for endophthalmitis after cataract surgery, Lohmann et al.7 and Therese et al.8 have shown a high positive rate in vitreous and aqueous samples subject to PCR analysis in cases of delayed-onset endophthalmitis. This is the first report on an explanted IOL and may suggest the possibility of P acnes colonies on the surface of an IOL. In our case, bacterial cultures of the conjunctival swab and the aqueous humor failed to demonstrate P acnes. Although we detected P acnes DNA by PCR in the explanted IOL, the possibility of contamination from the aqueous or the ocular surface during removal of the IOL cannot be excluded.
524
By SEM, a coating of biofilm-like aggregates, diphtheroid particles, and crystals were found on the surface of the IOL. The morphologies suggested a bacterial biofilm on the surface of the IOL,3,6 but the possibility of host cellular debris and melanosomes could not be excluded.6 In conclusion, we report the first detection of P acnes DNA on an explanted IOL in a case of chronic pseudophakic endophthalmitis. Morphologic study by SEM examination supports the possibility of P acnes colonies on the IOL. REFERENCES 1. Deramo VA, Ting TD. Treatment of Propionibacterium acnes endophthalmitis. Curr Opin Ophthalmol 2001; 12:225–229
J CATARACT REFRACT SURG - VOL 32, MARCH 2006
CASE REPORTS: PROPIONIBACTERIUM ACNES ON EXPLANTED IOL
2. Sawusch MR, Michels RG, Stark WJ, et al. Endophthalmitis due to Propionibacterium acnes sequestered between IOL optic and posterior capsule. Ophthalmic Surg 1989; 20:90–92 3. Busin M, Cusumano A, Spitznas M. Intraocular lens removal from eyes with chronic low-grade endophthalmitis. J Cataract Refract Surg 1995; 21:679–684 4. Teichmann KD. Propionibacterium acnes endophthalmitis requiring intraocular lens removal after failure of medical therapy. J Cataract Refract Surg 2000; 26:1085–1088 5. Hall GS, Pratt-Rippin K, Meisler DM, et al. Growth curve for Propionibacterium acnes. Curr Eye Res 1994; 13:465–466 6. Tanner V, Rosen P, Skinner AR, et al. Electron microscopy study of explanted intraocular lenses from clinically noninfected eyes. J Cataract Refract Surg 1998; 24:1145–1151 7. Lohmann CP, Linde H-J, Reischl U. Improved detection of microorganisms by polymerase chain reaction in delayed endophthalmitis after
8.
9.
10.
11.
cataract surgery. Ophthalmology 2000; 107:1047–1051; discussion by RN Van Gelder, 1051–1052 Therese KL, Anand AR, Madhavan HN. Polymerase chain reaction in the diagnosis of bacterial endophthalmitis. Br J Ophthalmol 1998; 82:1078–1082 Harada K, Tsuneyama K, Sudo Y, et al. Molecular identification of bacterial 16S ribosomal RNA gene in liver tissue of primary biliary cirrhosis: is Propionibacterium acnes involved in granuloma formation? Hepatology 2001; 33:530–536 Meisler DM, Mandelbaum S. Propionibacterium-associated endophthalmitis after extracapsular cataract extraction; review of reported cases. Ophthalmology 1989; 96:54–60; discussion by RE Smith, 60–61 Zambrano W, Flynn HW Jr, Pflugfelder SC, et al. Management of options for Propionibacterium acnes endophthalmitis. Ophthalmology 1989; 96:1100–1105
J CATARACT REFRACT SURG - VOL 32, MARCH 2006
525
Propionibacterium acnes DNA from an explanted intraocular lens detected by polymerase chain reaction in a case of chronic pseudophakic endophthalmitis Jui-Yang Lai, MS, Ko-Hua Chen, MD, Ying-Cheng Lin, MD, Wen-Ming Hsu, MD, Shui-Mei Lee, MD
We report a case of Propionibacterium acnes DNA from the explanted intraocular lens (IOL) a 76-year-old man with delayed-onset chronic endophthalmitis. The detection was by polymerase chain reaction (PCR). Slitlamp microscopic examination showed cells and flare in the anterior chamber, keratic precipitates on the endothelium, and multiple white plaques on both surfaces of the IOL. Propionibacterium acnes DNA was found in both the aqueous humor and the explanted IOL by PCR. Scanning electron microscopy showed diphtheroid particles, a coating of biofilm-like deposits, as well as scattered crystals on the surface of the explanted IOL and supported the possibility of P acne colonies on the IOL. J Cataract Refract Surg 2006; 32:522–525 Q 2006 ASCRS and ESCRS
Propionibacterium acnes infection is a main cause of delayed-onset chronic endophthalmitis associated with cataract surgery.1 The exact mechanisms by which the bacterium P acnes causes endophthalmitis after cataract surgery are not completely known. The binding of bacteria on the surface of the intraocular lens (IOL) during implantation, with its concealment between the IOL optic and capsular bags, is known to play a role in the pathogenesis of pseudophakic endophthalmitis.2 Evidence supporting this hypothesis comes from scanning electron microscopy
Accepted for publication July 11, 2005. From the Department of Chemical Engineering (Lai), National Tsing Hua University, Hsinchu, and the Division of Medical Engineering (Lai, Chen), National Health Research Institutes, Department of Ophthalmology (Chen, Lin, Hsu, Lee), Taipei Veterans General Hospital, National Yang Ming University (Chen, Hsu), Taipei, Taiwan, China. Supported by VGH-93-360-5 from the Taipei Veterans General Hospital. No author has a financial or proprietary interest in any material or method mentioned. Reprints requests to Ko-Hua Chen, MD, Department of Ophthalmology, Taipei Veterans General Hospital, 201 Shih-Pai Road, Section 2, Taipei, Taiwan 11217, China. E-mail: khchen@vghtpe. gov.tw. Q 2006 ASCRS and ESCRS Published by Elsevier Inc.
522
(SEM) morphologic studies.3 Positive cultures of P acnes came from the excised posterior capsular bag and explanted IOL,2,4 although the culture rate of P acnes was low.5 Furthermore, misinterpretation of IOL observations by SEM is possible.6 Polymerase chain reaction (PCR) has been shown to be a more sensitive method than culturing to detect intraocular P acnes infection in aqueous and vitreous samples from cases of delayed-onset endophthalmitis.7,8 However, PCR has not been used to detect P acnes on IOLs. Scanning electron microscopy has been used to demonstrate bacterial adherence to the IOL in studies of pseudophakic endophthalmitis.3,6 We report a case of delayed-onset chronic endophthalmitis and P acnes DNA from an explanted IOL detected by PCR. The images of the IOL surfaces on SEM examination support the possibility of bacterial adherence on the IOL. CASE REPORT A 76-year-old man presented to our department with progressive blurred vision, redness, tearing, and photophobia in the left eye. His history was remarkable for delayed-onset chronic endophthalmitis and secondary glaucoma after cataract surgery in the left eye since May 2001. In January 2001, he had cataract phacoemulsification surgery in the left eye, during which posterior capsule rupture occurred. An anterior chamber IOL (1-piece poly(methyl methacrylate) 4-point fixation, Alcon) was implanted. Before the 0886-3350/06/$-see front matter doi:10.1016/j.jcrs.2005.12.071
CASE REPORTS: PROPIONIBACTERIUM ACNES ON EXPLANTED IOL
surgery, cephalexin (250 mg every 6 hours) was prescribed for prophylaxis of infection. No antibiotics were added to the irrigation solution. Fifteen milligrams gentamicin subconjunctival injection was given postoperatively. Two months later, the left eye developed redness, pain, and blurred vision. Delayed-onset endophthalmitis was diagnosed by his surgeon, and in Febuary 2002, he was referred to our hospital for further management. On examination, the best corrected visual acuity (BCVA) in the left eye was 20/1200. Slitlamp microscopic examination demonstrated congestion of the left conjunctiva, mild stromal edema of the left cornea, cells and flare in the aqueous humor, keratic precipitates on the corneal endothelial layer, and white plaquelike deposits on both surfaces of the anterior chamber of the IOL (Figure 1). The posterior capsule was absent. After unsuccessful treatment with topical and systemic vancomycin and corticosteroids for 2 weeks, the patient had another surgery to remove the IOL in the anterior chamber and vitrectomy performed at our hospital. Before the surgery, the cultures from the conjunctiva and aqueous humor sample were taken. The 0.1 mL aqueous humor sample was sent for PCR detection of P acnes. An aqueous humor sample from a healthy cataract patient and a new IOL were used as the negative controls. After surgery, all signs and symptoms of endophthalmitis subsided. The BCVA improved to 20/40 with spectacles at 2 months follow-up. The explanted IOL was bisected and immediately submitted to the pathology laboratory in a dry, sterile container. Half the sample was sent for PCR, and the other half was immersed in Karnovsky fixation solution and processed for SEM (Model JSM 5600; Jeol). For SEM examination, the IOL optics were cut with a blade into 5, 2.0 mm31.5 mm31.0 mm3 pieces, which then were subjected to a series of fixation and dehydration treatments. Each piece was subsequently mounted onto an aluminum stub and coated with gold in a sputter coater. One piece was analyzed with an energy dispersive x-ray spectroscopy (Oxford Instruments America, Inc.) to define the nature of the crystals on the IOL.
Polymerase chain reaction was performed to amplify 16S rRNA gene fragments of P acnes. Antisense primer (50 -GGGTTGATAA(A/T)CCGCTTTCGCCTG-30 ) and sense primer (50 -GGGACACCCATCTCTGAGCAC-30 ) were designed specifically to amplify a 587 bp portion of the P acnes 16S rRNA gene (GenBank accession number PA16SLIP4).9 Using 5 mL extracted DNA samples, the following conditions were used for amplification: initial denaturation 94 C for 4 minutes and then 94 C for 30 seconds, 50 C for 30 seconds, and 72 C for 1 minute for 45 cycles. Alhough all cultures from the conjunctival swab, vitreous, and aqueous humor failed to demonstrate P acnes, P acnes DNA was detected from both the aqueous humor sample and the explanted IOL (Figure 2). Under SEM, a biofilm-like coating and scattered crystals were found spread over both sides of the optic surface from the explanted IOL (Figure 3, A). Diphtheroid particles with sizes equal to or smaller than approximately 0.6 mm and their morphologic characteristics were compatible with those in previous reports (Figure 3, B). It was noted that a few crystallike deposits were scattered on both surfaces of the IOL optics (Figure 3, C). From energy dispersive x-ray spectroscopic analyses, the crystal-like deposits were shown to be composed of sodium and chloride (Figure 3, D). DISCUSSION
Clinically, P acnes endophthalmitis manifested as a chronic iridocyclitis is characterized by granulomatousappearing keratic precipitates, hypopyon, and white plaques on the posterior capsule or IOL.10,11 The diagnosis of P acnes infection in pseudophakic endophthalmitis can be difficult because the culture results from the aqueous humor are usually negative due to the slow growth rate of M
1
2
3
4
5
500 bp-
Figure 1. Two weeks after treatment with systemic and topical vancomycin and corticosteroids, white plaque deposits on the IOL, cells and flare in the anterior chamber, and keratic precipitates on the endothelium were seen on by slitlamp microscopy.
Figure 2. Electrophoresis of PCR amplification products using primers for 1 587 bp portion of the P acnes 16S rRNA gene. Lane 1: 5 mL DNA extracted from the explanted IOL. Lane 2: Aqueous samples from the patient. Lane 3: Positive control. Lane 4: Aqueous humor from a healthy cataract eye as a negative control. Lane 5: A new IOL as a negative control. Marker (M) is a 100 bp ladder.
J CATARACT REFRACT SURG - VOL 32, MARCH 2006
523
CASE REPORTS: PROPIONIBACTERIUM ACNES ON EXPLANTED IOL
Figure 3. Scanning electron microscopic photographs showed the 3-dimensional view of the lens surface. A granule-like coating on the surface of the IOL was noted under lower manifestation (A). Upon higher manifestation, diphtheroid particles of !1 mm size were spread evenly over the whole surface (B), and scattered crystals were also noted (C). A: Original magnification, 500. B and C: Original magnification 2000. Energy dispersive x-ray spectroscopic analyses of the explanted IOL from this patient showed that sodium and chloride were distributed on the IOL surface (D). Note only carbon and oxygen on the optics and haptics of the control (data not shown).
P acnes5 and also possibly due to the sequestered state of the organisms in the remaining capsular bag.2 To increase the detection rate of P acnes responsible for endophthalmitis after cataract surgery, Lohmann et al.7 and Therese et al.8 have shown a high positive rate in vitreous and aqueous samples subject to PCR analysis in cases of delayed-onset endophthalmitis. This is the first report on an explanted IOL and may suggest the possibility of P acnes colonies on the surface of an IOL. In our case, bacterial cultures of the conjunctival swab and the aqueous humor failed to demonstrate P acnes. Although we detected P acnes DNA by PCR in the explanted IOL, the possibility of contamination from the aqueous or the ocular surface during removal of the IOL cannot be excluded.
524
By SEM, a coating of biofilm-like aggregates, diphtheroid particles, and crystals were found on the surface of the IOL. The morphologies suggested a bacterial biofilm on the surface of the IOL,3,6 but the possibility of host cellular debris and melanosomes could not be excluded.6 In conclusion, we report the first detection of P acnes DNA on an explanted IOL in a case of chronic pseudophakic endophthalmitis. Morphologic study by SEM examination supports the possibility of P acnes colonies on the IOL. REFERENCES 1. Deramo VA, Ting TD. Treatment of Propionibacterium acnes endophthalmitis. Curr Opin Ophthalmol 2001; 12:225–229
J CATARACT REFRACT SURG - VOL 32, MARCH 2006
CASE REPORTS: PROPIONIBACTERIUM ACNES ON EXPLANTED IOL
2. Sawusch MR, Michels RG, Stark WJ, et al. Endophthalmitis due to Propionibacterium acnes sequestered between IOL optic and posterior capsule. Ophthalmic Surg 1989; 20:90–92 3. Busin M, Cusumano A, Spitznas M. Intraocular lens removal from eyes with chronic low-grade endophthalmitis. J Cataract Refract Surg 1995; 21:679–684 4. Teichmann KD. Propionibacterium acnes endophthalmitis requiring intraocular lens removal after failure of medical therapy. J Cataract Refract Surg 2000; 26:1085–1088 5. Hall GS, Pratt-Rippin K, Meisler DM, et al. Growth curve for Propionibacterium acnes. Curr Eye Res 1994; 13:465–466 6. Tanner V, Rosen P, Skinner AR, et al. Electron microscopy study of explanted intraocular lenses from clinically noninfected eyes. J Cataract Refract Surg 1998; 24:1145–1151 7. Lohmann CP, Linde H-J, Reischl U. Improved detection of microorganisms by polymerase chain reaction in delayed endophthalmitis after
8.
9.
10.
11.
cataract surgery. Ophthalmology 2000; 107:1047–1051; discussion by RN Van Gelder, 1051–1052 Therese KL, Anand AR, Madhavan HN. Polymerase chain reaction in the diagnosis of bacterial endophthalmitis. Br J Ophthalmol 1998; 82:1078–1082 Harada K, Tsuneyama K, Sudo Y, et al. Molecular identification of bacterial 16S ribosomal RNA gene in liver tissue of primary biliary cirrhosis: is Propionibacterium acnes involved in granuloma formation? Hepatology 2001; 33:530–536 Meisler DM, Mandelbaum S. Propionibacterium-associated endophthalmitis after extracapsular cataract extraction; review of reported cases. Ophthalmology 1989; 96:54–60; discussion by RE Smith, 60–61 Zambrano W, Flynn HW Jr, Pflugfelder SC, et al. Management of options for Propionibacterium acnes endophthalmitis. Ophthalmology 1989; 96:1100–1105
J CATARACT REFRACT SURG - VOL 32, MARCH 2006
525