Cytomegalovirus as an etiologic factor in corneal endotheliitis.

PURPOSE: To investigate clinical manifestations and response to antiviral therapy of 8 patients with cytomegalovirus (CMV)-induced corneal endotheliitis who were diagnosed and treated at 2 university hospitals in Japan. DESIGN: Retrospective, consecutive, multicenter case series. PARTICIPANTS: Eight eyes of 8 patients diagnosed with active CMV corneal endotheliitis at Kyoto Prefectural University of Medicine and Ehime University School of Medicine. The diagnosis was made based on the detection by polymerase chain reaction assay of CMV, but not herpes simplex virus (HSV) and varicella zoster virus (VZV) DNA, in the aqueous humor from the affected eye. METHODS: Retrospective review of the clinical manifestations and responses to antiviral treatment. MAIN OUTCOME MEASURES: Patient profiles, including duration of corneal endotheliitis, systemic disease, intraocular pressure, and clinical manifestation of anterior and posterior segments. The clinical response to systemic and topical antiviral treatment was evaluated by slit-lamp examination. Corneal endothelial density was examined by specular microscopy. RESULTS: The average observation period after CMV detection was 10.4 months (range, 2-24 months). None of the patients had systemic immunodeficiency. Corneal manifestations included linear keratic precipitates associated with multiple coin-shaped lesions and local corneal stromal edema. Of the 8 patients, 4 had undergone penetrating corneal transplantation. Systemic ganciclovir therapy was used in 7 patients, and in 1 patient, valacyclovir was administered, with the corneal endotheliitis responding quickly to the early administration of galovir. At the final examination, 6 eyes had a clear cornea, but 2 eyes had bullous keratopathy. CONCLUSIONS: Besides HSV and VZV, CMV must be considered as an etiologic agent in patients with corneal endotheliitis. Cytomegalovirus corneal endotheliitis may be a newly identified clinical entity of reactivated CMV in the anterior chamber of individuals free of accompanying systemic symptoms.

Cytomegalovirus in aqueous humor from an eye with corneal endotheliitis.

PURPOSE: To report cytomegalovirus (CMV) DNA in aqueous humor from a patient with unilateral corneal endotheliitis. DESIGN: Case report. METHODS: A 51-year-old man presented with unilateral corneal endotheliitis with linear keratic precipitates and coin-shaped lesions. Tear and aqueous humor samples were subjected to polymerase chain reaction to look for DNA from herpes simplex virus (HSV), varicella zoster virus (VZV), and CMV. RESULTS: Aqueous humor from the diseased eye contained DNA from CMV but not HSV or VZV. Its specificity was confirmed by Southern blot tests. Intravenous ganciclovir treatment resulted in the localization of his corneal edema and the reduction in keratic precipitates. There was severe destruction of corneal endothelial cells. CMV DNA was not detected in tears or control samples. CONCLUSIONS: In this healthy man with corneal endotheliitis, we detected CMV DNA in aqueous humor from the affected eye, but not HSV or VZV. This suggests that CMV may cause corneal endotheliitis in patients without immunodeficiency.

The presence of Propionibacterium spp. in the vitreous fluid of uveitis patients with sarcoidosis.

PURPOSE: An immunological reaction to a bacterial antigen, such as Mycobacterium tuberculosis or Propionibacterium spp., is suspected to be an initial mechanism in the disorder known as sarcoidosis. We investigated whether or not P. acnes, P. granulosum or M. tuberculosis are present in the vitreous fluid of eyes suffering from uveitis with sarcoidosis. METHODS: Using polymerase chain reaction, we analysed the presence of P. acnes, P. granulosum and/or M. tuberculosis DNA in vitreous samples taken from six eyes with sarcoidosis and six control eyes. RESULTS: Among the six uveitis eyes with sarcoidosis, we detected P. acnes DNA in two eyes, P. granulosum DNA in four eyes, and both P. acnes and P. granulosum DNA in one eye, but no Propionibacterium spp. in the control eyes. M. tuberculosis DNA was not present in any of the patient or control eyes. CONCLUSIONS: This is the first report indicating the presence of Propionibacterium spp. and/or its DNA in the vitreous fluid of sarcoidic eyes with uveitis. This, therefore, supports the idea that Propionibacterium spp. are involved in the aetiology of uveitis in sarcoidosis.

Lidocaine inhibits tyrosine kinase activity of the epidermal growth factor receptor and suppresses proliferation of corneal epithelial cells.

BACKGROUND: Although lidocaine is recognized as an excellent topical corneal analgesic, its toxic effect on corneal epithelial cells limits its use during corneal epithelial wound healing. Mechanism of the impairment of corneal reepithelialization with lidocaine, however, has not been evaluated. The authors' previous study revealed that lidocaine inhibits the activity of tyrosine kinase receptors through the interaction with specific amino acid sequences around autophosphorylation sites, including acidic, basic, and aromatic amino acids. Epidermal growth factor receptor (EGFR), a tyrosine kinase receptor with an important role in epithelial cell proliferation after corneal wounding, also possesses these amino acids sequences around autophosphorylation sites. The authors hypothesized that lidocaine would suppress tyrosine kinase activity of EGFR and would impair corneal epithelial cell proliferation. METHODS: To investigate the effect of lidocaine (4 microM-40 mM) on epidermal growth factor (EGF)-stimulated autophosphorylation of EGFR, the authors studied purified EGFR in microtubes. They cultured human corneal epithelial cells (HCECs) with EGF and lidocaine to investigate the effect of lidocaine on cell proliferation and on autophosphorylation of EGFR in HCECs. RESULTS: Lidocaine (> or =400 microM) significantly suppressed EGF-stimulated autophosphorylation of the purified EGFR. In the HCEC study, EGF alone stimulated cell proliferation and increased autophosphorylation of EGFR in HCECs. Lidocaine (> or = 400 microM) significantly suppressed both the proliferation of HCECs promoted by EGF and EGF-stimulated autophosphorylation of EGFR. CONCLUSION: Lidocaine directly inhibits tyrosine kinase activity of EGFR and suppresses the corneal epithelial cell proliferation.

Tight junction-related protein expression and distribution in human corneal epithelium.

PURPOSE: To investigate the expression and cellular distribution of the tight junction-related proteins occludin, claudin and ZO-1 in human corneal epithelium. METHODS: Light and electron immunohistochemistry was used to determine tissue distribution of occludin, claudin-1 and ZO-1 in the human corneal epithelium. Reverse transcription-polymerase chain reaction was used to reveal claudin mRNA expression in human corneal epithelium. RESULTS: In transverse sections, occludin and ZO-1 were localized at the apical cell-cell junctions between superficial cells in stratified corneal epithelium. Both basal and basolateral membranes of superficial cells were stained by the claudin-1 antibody, but no apical membrane staining was observed. In en face sections, claudin-1 and ZO-1 antibodies showed as bands that corresponded to the junctional complex. Claudin-1 staining of superficial cell cytoplasm was also observed. Occludin staining was seen at the junctional complex, where it was not continuous, but dotted along the cell junctions. The transcripts for claudin-1 and several other claudin isotypes, such as -2, -3, -4, -7, -9 and -14 were identified. CONCLUSION: Not only occludin, but also some claudins act as integral transmembrane proteins in the corneal epithelium. ZO-1 is a component of the corneal epithelial tight junction, as it is in most epithelial cells.

Comparison of ultrastructure, tight junction-related protein expression and barrier function of human corneal epithelial cells cultivated on amniotic membrane with and without air-lifting.

PURPOSE: To evaluate the usefulness of the air-lifting technique for culturing corneal limbal epithelial cells on amniotic membrane (AM) for use in ocular surface reconstruction. A cultured sheet that has a good barrier function should be better for this purpose. In corneal epithelium, tight junctions (TJ) play a vital role in the barrier function. The TJ complex includes the integral transmembrane proteins occludin and the claudins, and some membrane-associated proteins such as ZO-1. In this paper, we investigated the barrier function and the expression of TJ related proteins. METHODS: Corneal limbal epithelium obtained from donor corneas and cultivated on acellular AM was divided into two groups. These were the non-air-lifting (Non-AL) group, which was continuously submerged in medium, and the air-lifting (AL) group, which was submerged in medium for 3 weeks, then exposed to air by lowering the medium level. Morphology and the permeability to horseradish peroxidase (HRP) were determined by electron microscopy. Tight junction (TJ)-related protein and mRNA expression changes were assessed by immunoblotting and reverse transcription-polymerase chain reaction. RESULTS: The cultures of both groups formed 4-5-layer-thick, well-stratified epithelium. The AL cultures had tightly packed epithelial cells with all the HRP/diaminobenzidine (DAB) reaction product accumulated on the apical surface of the superficial cells. The Non-AL culture, by contrast, had more loosely packed epithelial cells with larger intercellular spaces. The HRP/DAB reaction product penetrated the intercellular space to a depth of 3-4 cell layers. Statistically, there was a significant difference in intercellular spaces and desmosome count in the superficial cells between the groups. With AL, TJ-related proteins localized at the apical portion of the lateral membrane. TJ-related protein and mRNA amounts were not changed by AL while claudin subtype expression became more consistent and closer to that of in vivo corneal epithelium. CONCLUSIONS: The AL technique reduces intercellular spaces in the superficial cells and promotes the formation of the barrier function. It is useful in culturing corneal epithelial cells for use in ocular surface reconstruction.

[Comparative studies on activities of antimicrobial agents against causative organisms isolated from patients with urinary tract infections (2000). I. Susceptibility distribution].

The bacterial strains isolated from patients diagnosed as having urinary tract infections (UTIs) in 10 institutions in Japan were supplied between the period of August 2000 and July 2001. Then, the susceptibilities of them to many kinds of antimicrobial agents were investigated. The number of them were 511 strains. The breakdown of these strains was Gram-positive bacteria as 29.0% and Gram-negative bacteria as 71.0%. Susceptibilities of these bacteria to antimicrobial agents were as follows; vancomycin (VCM), ampicillin (ABPC) and imipenem (IPM) showed strong activities against Enterococcus faecalis. No increase in low-susceptible strains of E. faecalis observed against these antimicrobial agents. VCM showed a strong activity against MRSA preventing growth of all strains with 1 microgram/ml. In addition, the activity of arbekacin (ABK) was strong with the MIC90 of 2 micrograms/ml against MRSA and prevented growth of all strains with 4 micrograms/ml. ABK showed a strong activity against Staphylococcus epidermidis preventing growth of all strains with 0.5 microgram/ml. ABPC, cefotiam (CTM) and cefozopran (CZOP) also showed a relatively strong activity against S. epidermidis (MIC90: 4 to 8 micrograms/ml). Against Escherichia coli, carbapenems showed high activities: meropenem (MEPM) prevented growth of all strains within 0.125 microgram/ml; IPM prevented growth of all strains with 0.25 microgram/ml. CZOP and CTM also showed strong activities against E. coli: MIC90 of CZOP was within 0.125 microgram/ml; MIC80 and MIC90 of CTM were 0.25 and 0.5 microgram/ml, respectively. Quinolone resistant E. coli was detected at frequency of 14.0%, which was significantly higher than that in the last year. Almost all drugs showed strong activities against Klebsiella pneumoniae and Proteus mirabilis, and MEPM prevented growth of all strains within 0.125 microgram/ml. Against Pseudomonas aeruginosa, almost drugs were not so active. The MIC90 of carbapenems and gentamicin (GM) were 16 micrograms/ml and those of all other drugs were more than 32 micrograms/ml. Against Serratia marcescens, the MIC90 of IPM and GM were the lowest value being 2 micrograms/ml, and that of MEPM was 4 micrograms/ml.

[Comparative studies on activities of antimicrobial agents against causative organisms isolated from patients with urinary tract infections (2000). II. Background of patients].

Five-hundred eighty eight bacterial strains isolated from 435 patients diagnosed as having urinary tract infections (UTIs) in 10 institutions in Japan were supplied between August 2000 and July 2001. Then, the clinical background of patients were investigated such as sex, age, and type of infections, infections and kind of bacteria, frequency of bacteria isolation by age and infections, bacteria and infections by timing of antibiotics administration, and bacteria and infections by surgical procedures. About the relationship between age and sex of patients and type of infections, the number of male patients aged less than 50 years was few, and complicated UTIs without indwelling catheter was the most frequent. In females, the number of patients aged less than 20 years was few. The majority of female patients aged 40 years and over had complicated UTIs while uncomplicated UTIs was most frequent in the patients being twenties. As for type of infections and kind of bacteria, Escherichia coli decreased when the infections became complicated, and pseudomonas aeruginosa increased when the infection became complicated. Enterococcus faecalis was isolated more frequently in complicated UTIs than in uncomplicated UTIs. Considering this result by age of patients, isolated frequency of E. coli was gradually decreased with aging in patients aged 20 years and over with uncomplicated UTIs or complicated UTIs without indwelling catheter. The isolated frequencies of Klebsiella spp., P. aeruginosa, and E. faecalis were high in the patients with complicated UTIs without indwelling catheter. In the patients aged 70 years and over with complicated UTIs with indwelling catheter, P. aeruginosa and E. faecalis were frequently isolated. As for type of causative organisms in UTIs before and after the administration of antibiotics, the isolation of bacteria was remarkably decreased after administration in the patients with uncomplicated UTIs and complicated UTIs without indwelling catheter. E. coli decreased after administration of antibiotics, and P. aeruginosa increased after administration in the patients with all types of infections. As for type of causative organisms in UTIs and surgical procedures, E. coli was frequently isolated in the patients without surgery in all types of infections, while P. aeruginosa was frequently isolated in the patients who underwent surgery. In uncomplicated UTIs, Proteus spp. and E. faecalis were frequently isolated in the patients with surgery. In complicated UTIs without indwelling catheter, Klebsiella spp. was frequently isolated in the patients without surgery and E. faecalis was frequently isolated in the patients with surgery. In complicated UTIs with indwelling catheter, most of organisms except P. aeruginosa and S. aureus were frequently isolated in the patients without surgery.

The association of HLA with young-onset keratoconus in Japan.

PURPOSE: To report the association of HLA antigens with keratoconus in Japanese patients. DESIGN: Observational consecutive case series. METHODS: In 90 consecutive Japanese keratoconus patients, HLA class I (HLA-A, -B, -C) and class II (HLA-DR, -DQ) were analyzed. RESULTS: Compared with control frequencies, based on mean gene frequencies for the Japanese population, higher frequencies of HLA-A26, B40, and DR9 antigens were found in patients whose conditions were diagnosed before 20 years of age (chi(2) = 6.45, P =.01; chi(2) = 6.78, P =.01; chi(2) =3.99, P =.05, respectively), but were not found in patients whose conditions were diagnosed later in life. Men were significantly younger at diagnosis than were women. No obvious relation was found between HLA antigens and other clinical data. CONCLUSION: HLA-A26, B40, and DR9, which were found relatively frequently in the ancient Japanese population, seem to be associated with keratoconus in younger individuals.

[Comparative studies on activities on antimicrobial agents against causative organisms isolated from patients with urinary tract infections (2000). III. Secular changes in susceptibility].

The bacteria (Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa) isolated from patients diagnosed as having urinary tract infections (UTIs) in 10 institutions in Japan were supplied between August 2000 and July 2001. Then, the susceptibilities of these bacteria to various antimicrobial agents were examined, and the results were compared with those obtained between 1992 and 1999. Comparison was made by classifying strains isolated from patients into those in uncomplicated UTIs and those in complicated UTIs (including with or without indwelling catheter). E. faecalis showed good susceptibility to ampicillin (ABPC) and imipenem (IPM), and the MIC90s were 2 micrograms/ml. Also, E. faecalis showed good susceptibility to vancomycin (VCM). However, the MIC90, which was 2 micrograms/ml between 1992 and 1999, rose to 4 micrograms/ml in patients with complicated UTIs because the strains inhibited at 4 micrograms/ml increased more than before. The low susceptibility of S. aureus to arbekacin (ABK) in complicated UTIs, as shown in 1998 and 1999, recovered in 2000, and no strains inhibited at > or = 4 micrograms/ml were detected. E. coli showed good susceptibility to CTM (MIC90: 0.25-0.5 microgram/ml) and CZOP (MIC90: < or = 0.125 microgram/ml) and was not resistant to those. E. coli also showed good susceptibility to the other drugs except to penicillins. Decreases in susceptibility of E. coli to quinolones, ciprofloxacin (CPFX), and sparfloxacin (SPFX) were observed in the patients with complicated UTIs. The susceptibility of Klebsiella spp. to all drugs did not significantly change in 2000 and was generally good except to penicillins. Although the susceptibility of P. aeruginosa to carbapenems was notable, the MIC90 went up from 4 micrograms/ml to 16 micrograms/ml in complicated UTIs compared with those observed in the previous year.

Two sporadic cases of infections due to Klebsiella pneumoniae resistant to expanded-spectrum cephalosporins in Japan.

TEM- or SHV-type extended-spectrum beta-lactamases (ESBLs) are of clinical concern in Europe and the United States, whereas bacterial strains producing such types of ESBLs have not been reported in Japan. We report here two cases of infection due to Klebsiella pneumoniae resistant to extended-spectrum cephalosporins in Japan. A ceftadizime-resistant K. pneumoniae strain (minimum inhibitory concentration; 32 &mgr;g/ml) was isolated transiently from the sputum of an 87-year-old woman with acute myocardial infarction and pneumonia (patient 1). Ceftadizime-susceptible and -resistant (minimum inhibitory concentration; >/=8 &mgr;g/ml) K. pneumoniae strains were isolated over a month from the blood, ascites, and feces of a 44-year-old man after bone marrow transplantation for acute lymphoblastic leukemia (patient 2); this patient died of K. pneumoniae sepsis and peritonitis followed by multiple organ failure. These isolates produced penicillinase, which was inhibited by clavulanic acid. A polymerase chain reaction (PCR) study showed that both isolates carried the SHV or LEN genes, but not the TEM, Toho-1, and IMP-1 genes. The pulsed-field gel electrophoresis profile of the strain isolated from patient 1 was genetically distinguishable from the profiles of the strains isolated from patient 2. It appeared that mutation of the beta-lactamase gene may have occurred in the body of patient 2, since the genotypes of the ceftadizime-susceptible and -resistant isolates from this patient were identical. Another 12 strains of K. pneumoniae, isolated from other patients in the same wards during the period in which the K. pneumoniae strains were isolated from patients 1 and 2, did not produce ESBLs and showed different genotypes. The results suggest that these isolates of resistant K. pneumoniae did not spread by cross transmission in the hospital and that the two cases were sporadic. Surveillance of these types of resistant bacteria is necessary, since they may well be present in other hospitals in Japan. Although the organisms are suspected to produce SHV-type ESBLs or LEN-1 variant beta-lactamases, further studies are necessary to specify the resistance genes.