Applying Metagenomic Analysis Using Nanopore Sequencer (MinION) for Precision Medicine in Bacterial Keratoconjunctivitis: Comprehensive Validation of Molecular Biological and Conventional Examinations.

Smear microscopic examination and culture of the corneal scrapings are the gold standards for the diagnosis of bacterial keratoconjunctivitis. High-sensitivity molecular biological examinations of the ocular surface specimens are used clinically. However, the results require careful interpretation to avoid the unintentional detection of indigenous bacteria. Results of conventional and state-of-the-art examinations require clinical verification for specificity and sensitivity. In this study, smear microscopic examination, culture, and nanopore sequencing using the MinION of ocular surface specimens from eight clinically diagnosed bacterial keratoconjunctivitis cases were performed and compared. Seven of the eight cases (87.5%) were smear positive and five (62.5%) were culture positive. The former showed the same genus in >60% of the classified reads as one specific bacterium inferred from the smear microscopy when sequenced by the MinION. In two of the three culture-negative cases, the smear-positive images were highly reminiscent of the species comprising most of the MinION sequences. Four of the five culture-positive cases were consistent with the most prevalent bacteria in the sequencing results. Probable contamination among specimens processed on the same day were observed. In conclusion, the microscopic examination of the corneal scraping specimens may be more sensitive and specific than the culture examination. Additionally, although metagenomic analysis using the MinION contributes to more precise medication for bacterial keratoconjunctivitis, contamination can affect the results.

Severe Ocular Complications Associated With Wearing of Contact Lens in Japan.

OBJECTIVES: To examine the severe ocular complications associated with contact lens wearing in Japan. METHOD: A questionnaire was sent to 964 ophthalmologist training facilities inquiring for cases of contact lens-associated complications from April 2016 to March 2018. The inclusion criteria were as follows: (1) corrected distance visual acuity ≤0.1 decimal after treatment for 3 months, (2) corneal perforation observed during follow-up, and (3) requiring surgery. A secondary analysis was conducted, inquiring for further information on the type of contact lens, clinical manifestations, and course of treatment. RESULTS: Forty-two patients with infectious keratitis met the inclusion criteria. Eight patients were users of rigid gas-permeable contact lens, and 34 were users of soft contact lens. Microbiological tests were positive in 73.0%. The organisms isolated in microbiological culture were bacteria in 11 patients (Pseudomonas aeruginosa in 9 patients), fungi in 2 patients, and Acanthamoeba in 14 patients. Ten patients were treated with local antibiotics, 11 with a combination of systemic antibiotics, and 21 with a combination of surgical approaches, including 13 with corneal transplantation. CONCLUSIONS: The major cause of serious contact lens-associated ocular complications was microbial keratitis, and P. aeruginosa and Acanthamoeba were the major pathogens in Japan.

Orbital abscess caused by Exophiala dermatitidis following posterior subtenon injection of triamcinolone acetonide: a case report and a review of literature related to Exophiala eye infections.

BACKGROUND: Subtenon injection of triamcinolone acetonide (STTA) has been widely adopted in the clinical setting of ophthalmology and its infectious complications are rare. However, orbital abscess following STTA has been reported in seven cases. Furthermore, although eye infections due to Exophiala species are uncommon, there have been 19 cases to date. E. jeanselmei, E. phaeomuriformis, E. werneckii, and E. dermatitidis have been reported to cause human eye infections; however, to the best of our knowledge, orbital abscess caused by E. dermatitidis has not yet been reported. We describe the first documented case of fungal orbital abscess caused by E. dermatitidis following STTA. We also review the related literature of orbital abscess following STTA, as well as eye infections caused by the four Exophiala species. CASE PRESENTATION: The patient was a 69-year-old Japanese woman with diabetic mellitus. She had a macular oedema in her right eye, which occurred secondary to branch retinal vein occlusion. An orbital abscess caused by E. dermatitidis occurred 4 months after the second STTA for the macular oedema, which was successfully treated by a surgical debridement and systemic administration of voriconazole. CONCLUSIONS: Our findings in the patient and from our literature survey caution ophthalmologists to the fact that STTA can cause fungal orbital infections, especially in diabetic patients. Furthermore, surgical treatment is one of the most important risk factors.

Microsporidial keratitis retrospectively diagnosed by ultrastructural study of formalin-fixed paraffin-embedded corneal tissue: a case report.

BACKGROUND: The utility of formalin-fixed paraffin-embedded (FFPE) corneal tissue specimens for retrospective diagnosis of microsporidial keratitis was evaluated by transmission electron microscopy (TEM) analysis and the possible second case of microsporidial keratitis after Descemet stripping automated endothelial keratoplasty (DSAEK) was described. CASE PRESENTATION: A 68-year-old man presented with multiple crystalline opacities in the corneal stroma that progressed extremely slowly after DSAEK. Fungiflora Y staining of corneal scrapings from the affected regions revealed an oval microorganism. Topical voriconazole administration was ineffective and penetrating keratoplasty was performed. Histological and molecular analyses were carried out on the excised cornea. Ziehl-Neelsen staining revealed an acid-fast, oval organism that was visible by ultraviolet illumination after Fungiflora Y and Uvitex 2B staining, whereas periodic acid-Schiff and Grocott's staining did not yield any significant findings. Microsporidium was detected by TEM of FFPE tissue. Nosema or Vittaforma sp. was suspected as the causative microorganism by PCR of FFPE tissue and by the fact that those species are known to cause eye infection. The corneal graft has maintained transparency at 1 year and half postoperatively. CONCLUSIONS: This is the first known case of microsporidial keratitis diagnosed retrospectively by molecular and ultrastructural study of FFPE tissue, and the possible second case of microsporidial keratitis after DSAEK. Microsporidial keratitis should be considered when corneal opacity refractory to conventionally known therapy would occur after DSAEK. Our findings suggest that more microsporidial keratitis cases than have been reported to date can be identified by TEM or PCR examination of FFPE corneal specimens.

Human CD31 on porcine cells suppress xenogeneic neutrophil-mediated cytotoxicity via the inhibition of NETosis.

BACKGROUND: Xenotransplantation is one of the promising strategies for overcoming the shortage of organs available for transplant. However, many immunological obstructions need to be overcome for practical use. Increasing evidence suggests that neutrophils contribute to xenogeneic cellular rejection. Neutrophils are regulated by activation and inhibitory signals to induce appropriate immune reactions and to avoid unnecessary immune reactivity. Therefore, we hypothesized that the development of neutrophil-targeted therapies may have the potential for increased graft survival in xenotransplantation. METHODS: A plasmid containing a cDNA insert encoding the human CD31 gene was transfected into swine endothelial cells (SEC). HL-60 cells were differentiated into neutrophil-like cells by culturing them in the presence of 1.3% dimethyl sulfoxide for 48 hours. The cytotoxicity of the differentiated HL-60 cells (dHL-60) and peripheral blood-derived neutrophils was evaluated by WST-8 assays. To investigate the mechanism responsible for hCD31-induced immunosuppression, citrullinated histone 3 (cit-H3) and phosphorylation of SHP-1 were detected by a cit-H3 enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. RESULTS: A significant decrease in dHL-60 and neutrophil-mediated cytotoxicity in SEC/hCD31 compared with SEC was seen, as evidenced by a cytotoxicity assay. Furthermore, the suppression of NETosis and the induction of SHP-1 phosphorylation in neutrophils that had been co-cultured with SEC/CD31 were confirmed by cit-H3 ELISA and Western blotting with an anti-phosphorylated SHP-1. CONCLUSION: These data suggest that human CD31 suppresses neutrophil-mediated xenogenic cytotoxicity via the inhibition of NETosis. As CD31 is widely expressed in a variety of inflammatory cells, human CD31-induced suppression may cover the entire xenogeneic cellular rejection, thus making the generation of human CD31 transgenic pigs very attractive for use in xenografts.

Isolates and antibiotic susceptibilities of endogenous bacterial endophthalmitis: A retrospective multicenter study in Japan.

Endogenous bacterial endophthalmitis, also called metastatic endophthalmitis, is a rare bacterial endophthalmitis derived from distant infectious foci via the bloodstream. This infection can potentially cause not only severe visual disturbance, but also loss of the eyeball or death, as most patients are immunocompromised. This retrospective Japanese multicenter study analyzed 32 eyes in 25 definitive cases. Twelve patients (48.0%) had diabetes mellitus. Typical ocular findings were vitreous haze (87.5%), cells in the anterior chambers (62.5%) and retinal infiltrates (50.0%). Elevated body temperature (64.0%), high serum C-reactive protein (96.0%) and leukocytosis (52.0%) were also frequently observed. Culture positivity rates for intraocular fluid were higher in the vitreous (62.5%) versus aqueous humor (28.6%). High positivity rates were also observed for blood (57.1%) and central venous catheters (100%). The most common pathogen was Staphylococcus aureus (10 cases), including methicillin-resistant S. aureus (4 cases). The next most common pathogen was Klebsiella pneumoniae (7 cases), which was highly associated with liver abscess. Compared to a previous 1991 national multicenter study, there has been a fourfold increase in the ratio of S. aureus. Antibiotic susceptibility tests revealed that all Gram-positives were susceptible to vancomycin and all Gram-negatives were susceptible to third-generation cephalosporins, imipenem/cilastatin, gentamycin and levofloxacin. Prognostic factors influencing poor visual outcome included poor initial visual acuity (p < 0.01), K. pneumoniae (p = 0.027) and gram-negative bacteria (p = 0.014) as the causative bacteria. Intravitreal antibiotic injection in combination with vancomycin and ceftazidime may be applicable for use as part of the standard treatment regimen for EBE.

Quantification of Eccentric Fixation Using Spectral-Domain Optical Coherence Tomography.

PURPOSE: This study aimed to evaluate the utility of optical coherence tomography (OCT) in the quantification of eccentric fixation in amblyopic patients. MATERIAL AND METHODS: In this study, 14 amblyopic patients and 10 healthy volunteers were enrolled. Under non-mydriatic conditions, fixation tests were performed directly using a fixation ophthalmoscope and indirectly using spectral-domain OCT. For evaluations using OCT, the distance between the fovea and the fixation point, which was determined by a cross-sectional image, was measured. RESULTS: On evaluations of healthy volunteers by OCT, the mean distance between the fixation point and the fovea was 80.4 ± 37.7 µm for the dominant eyes and 63.7 ± 36.4 µm for non-dominant eyes (p = 0.41). In amblyopic patients, on evaluation by OCT, the mean distance between the fixation point and the fovea was 193.8 ± 188.3 µm in amblyopic eyes and 83.5 ± 39.3 µm in paired fellow eyes (p = 0.02). Although OCT could detect eccentric fixation points in all the affected eyes of amblyopic patients, fixation ophthalmoscope was unable to quantify them in 2 of 14 affected eyes. CONCLUSIONS: Compared with a fixation ophthalmoscope, our method using OCT seems to be superior both in quantification and detection of eccentric fixation in amblyopic patients, without the need for mydriasis.

Human CD200 suppresses macrophage-mediated xenogeneic cytotoxicity and phagocytosis.

PURPOSE: Various strategies, such as the generation of alpha-1,3-galactosyltransferase knocked-out pigs and CD55 transgenic pigs, have been investigated to inhibit pig to human xenogeneic rejection. Our aim is to develop strategies to overcome the hurdle of not only hyper acute rejection, but also that of cellular xenogeneic rejection (CXR). Although macrophages have been well known to play a critical role in CXR, monocyte/macrophage-mediated xenogeneic rejection has not been well studied. In this study, we evaluated the effect of CD200 in xenogeneic rejection by macrophages. METHODS: Naïve swine endothelial cells (SEC) and SEC/CD200 were co-cultured with M0 macrophages and the cytotoxicity was measured by a WST-8 assay. The phagocytosis of SEC and SEC/CD200 by macrophages was analyzed by flow cytometry. RESULTS: While CD200 failed to suppress a significant amount of cytotoxicity against SEC by monocytes, M0 macrophage-mediated cytotoxicity was significantly suppressed by human CD200. The phagocytosis by M0 macrophages was also tested. The phagocytosis assay revealed that human CD200 suppresses M0 macrophage-mediated phagocytosis. CONCLUSIONS: Our findings indicate that human CD200 suppresses the xenogeneic rejection by CD200R+ macrophages and that the generation of hCD200 transgenic pigs for use in xenografts is very attractive for preventing the macrophage-mediated rejection.

Correction to: Human CD200 suppresses macrophage-mediated xenogeneic cytotoxicity and phagocytosis.

In the original publication, the fifth author name was erroneously published as "Patmika Jiaravuthiasan". The correct author name should read as, "Patmika Jiaravuthisan". The original article was corrected.

Corneal Erosion With Pigments Derived From a Cosmetic Contact Lens: A Case Report.

PURPOSE: To report a case of corneal erosion with pigments derived from a cosmetic contact lens (CCL). METHODS: A 29-year-old woman complained of foreign body sensation after wearing CCL. Slit-lamp examination of her left eye revealed corneal erosion with yellow pigment at its edges. The surface of the CCLs was examined using the rub-off test and scanning electron microscopy (SEM). An elementary analysis using SEM with energy-dispersive x-ray (SEM-EDX) was also performed. RESULTS: On the back surface of the CCL, the pigments easily came off during the rub-off test. An SEM examination found exposed pigments with needle-like construction on the back surface. SEM-EDX revealed the presence of carbon, oxygen, iron, titanium, and chlorine in the pigments. CONCLUSION: Direct contact between the bare pigments on a CCL and the corneal surface can cause corneal damage.

A super-infection in the cornea caused by Stemphylium, Acremonium, and α-Streptococcus.

BACKGROUND: Polymicrobial keratitis with fungus and bacteria can lead to blindness and is challenging to treat. Here, we introduce a case of fungal keratitis caused by two different strains in addition to definite bacterial super-infection caused by an α-Streptococcus sp., and describe the importance of microscopic examination. CASE PRESENTATION: A 74-year-old woman, who had a past history of infection with leprosy, presented with conjunctival hyperaemia, pain, and corneal opacity in her right eye. Under the presumptive diagnosis of infectious keratitis, corneal scrapings were stained by various reagents and inoculated on several agar plates. Microscopic findings of the scrapings revealed fungi and a small number of Gram-positive cocci. Multiple anti-fungal therapies with levofloxacin ophthalmic solution were administered. Although empiric treatment was initially effective, keratitis recurred 10 days after its initiation. Repeated corneal scraping revealed an abundance of Gram-positive chain cocci and a small amount of fungi, resulting in the switching of an antibiotic medication from levofloxacin to moxifloxacin and cefmenoxime. Keratitis resolved gradually after the conversion. Stemphylium sp., Acremonium sp., and α-Streptococcus sp. were simultaneously isolated from the corneal scrapings. CONCLUSIONS: To the best of our knowledge, this is the first case of fungal keratitis caused by Stemphylium sp., and also the first case of super-infection in the cornea caused by two different fungi and one bacterium. Microscopic examination of the corneal scrapings was beneficial in rapid decision of changing to appropriate drug according to the dominancy of pathogenicity.

Pathological Analysis of Ocular Lesions in a Murine Model of Sjögren's Syndrome.

Sjögren's syndrome (SS) is a systemic autoimmune disease characterized by severe inflammation of exocrine glands such as the salivary and lacrimal glands. When it affects the lacrimal glands, many patients experience keratoconjunctivitis due to severely dry eyes. This study investigated the pathological and immunological characteristics of ocular lesions in a mouse model of SS. Corneal epithelial injury and hyperplasia were confirmed pathologically. The number of conjunctival mucin-producing goblet cells was significantly decreased in the SS model mice compared with control mice. Expression levels of transforming growth factor (TGF)-ß, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and C-X-C motif chemokine (CXCL) 12 were significantly higher in the corneal epithelium of the SS model mice than in control mice. Inflammatory lesions were observed in the Harderian, intraorbital, and extraorbital lacrimal glands in the SS model mice, suggesting that the ocular glands were targeted by an autoimmune response. The lacrimal glands of the SS model mice were infiltrated by cluster of differentiation (CD)4⁺ T cells. Real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed significantly increased mRNA expression of TNF-α, TGF-ß, CXCL9, and lysozyme in the extraorbital lacrimal glands of the SS model mice compared with control mice. These results add to the understanding of the complex pathogenesis of SS and may facilitate development of new therapeutic strategies.

Intraoperative optical coherence tomography (RESCAN® 700) for detecting iris incarceration and iridocorneal adhesion during keratoplasty.

There is no report focusing on the visualization of the iris incarceration or the iridocorneal adhesion during keratoplasty by use of microscope-integrated intraoperative optical coherence tomography (MIOCT). The purpose of this study is to report the usefulness of MIOCT for detecting iris incarceration and iridocorneal adhesions during penetrating keratoplasty (PK) and deep anterior lamellar keratoplasty (DALK). MIOCT system was applied both in a patient who underwent PK for corneal leukoma and in a patient who underwent DALK for keratoconus. During the surgeries, we obtained cross-sectional images around the host-graft interface by operating the foot switch of microscope without discontinuing the surgical procedure. Intraoperative MIOCT findings and postoperative outcomes were examined. An iris incarceration at the host-graft interface was visualized during surgery after corneal suture in PK, which allowed surgeons to return the iris to its original position instantly. In DALK, misdirected air into the posterior chamber could also be seen at the end of the DALK. This iridocorneal adhesion was resolved by fluid injection through paracentesis. Secondary glaucoma and graft rejection have not occurred postoperatively in both cases. The MIOCT system provides advantages such as prevention of secondary glaucoma and rejection following PK and DALK.

Corynebacterium lowii sp. nov. and Corynebacterium oculi sp. nov., derived from human clinical disease and an emended description of Corynebacterium mastitidis.

Strains of members of the genus Corynebacterium derived from ophthalmologic patients in Japan, Belgium and Switzerland and found to be closely related to-, but distinguishable from Corynebacterium mastitidis by 16S rRNA gene sequencing, were characterized using biochemical, chemotaxonomic, MALDI-TOF mass spectrometry and antimicrobial susceptibility methods and DNA-DNA hybridization as well as by whole-genome sequencing (WGS). Based on this investigation, we describe Corynebacterium lowii sp. nov. and Corynebacterium oculi sp. nov., derived from human ocular specimens, as well as emend the description of Corynebacterium mastitidis. Type strains for these species are: C. lowii R-50085T (=LMG 28276T =CCUG 65815T) and C. oculi R-50187T (=LMG 28277T =CCUG 65816T). DNA G+C content was found to be 62.2 % (by HPLC) and 62.8 % (by WGS) for C. lowii R-50085T, 64.1 % (HPLC) and 64.8 % (WGS) for C. oculi R-50187T and 67.8 % (HPLC) for C. mastitidis LMG 19040T [=S-8T =CCUG 38654T =CECT 4843T =CIP 105509T =DSM 44356T =IFO (NBRC)16160T =JCM 12269T].

[Multicenter Prospective Observational Study of Fungal Keratitis--Identification and Susceptibility Test of Fungi].

PURPOSE: To investigate the causative fungi of fungal keratitis in Japan and their drug susceptibility. METHODS: Identification and antifungal susceptibility test for 8 drugs (micafungin, amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, miconazole and pimaricin) were performed using isolated fungi from patients with fungal keratitis treated at 27 facilities in Japan between November 1, 2011 and October 31, 2013. RESULTS: Fungal strains were detected in 72 (50.7%) out of 142 samples. The major isolates were Fusarium spp. (18), Candida parapsilosis (12), C. albicans (11) and Alternaria spp. (6), in all, fungi of 31 species were identified by gene analysis. In the yeast-like fungi, susceptibility rates were evident for more than 80% in voriconazole, pimaricin, flucytosine, micafungin, amphotericin B and fluconazole. In filamentous fungi, the susceptibility rate was less than 50% except for PMR (90%). Fusarium spp., which were susceptible to amphotericin B and pimaricin, showed lower susceptibility rates compared with other genera. CONCLUSIONS: Although various genera and species of fungi cause fungal keratitis, the obtained drug susceptibility data in this study demonstrates the different susceptibility patterns among the major isolates (Fusarium spp., C. parapsilosis, C. albicans and other groups). This is important evidence useful for fungal keratitis treatment.

[Multicenter Prospective Observational Study of Fungal Keratitis--Current Status of Patients' Background, Clinical Findings, Treatment and Prognosis].

PURPOSE: To investigate the current status of fungal keratitis in Japan. METHODS: The patients with fungal keratitis were examined at 27 facilities in Japan from November 1st 2011 to October 31st 2013, concerning isolates, patient background, clinical findings, treatment and prognosis. RESULTS: Out of 139 cases, 133 were diagnosed as fungal keratitis, of which fungi were isolated from 72 samples of 71 cases (yeast-like fungi 32 strains and filamentous fungi 40 strains). The corrected visual acuity at the first visit of 88 cases (66.2%) was less than 20/200 and 42 cases (31.6%) were involved with deep stromal lesions, indicating high proportion of severe cases in this study. Three months later, 56 cases (42.1%) were still under treatment, and corrected visual acuity of 57 cases (42.9%) was less than 20/200. In cases with yeast-like fungi, there were significantly more cases with past history of corneal diseases, ocular surgery including keratoplasty, and eye drops' use such as steroids than those with filamentous fungi. On the other hand, there were significantly more cases of filamentous fungi, with trauma on the onset and with intervention of previously attending doctors than those with yeast-like fungi. Logistic regression analyses revealed that contact lens wearing was a significant factor of good prognosis, and yeast-like fungi as one of poor outcome compared with no fungal isolation. CONCLUSION: Although the choice of antifungal drugs has been increasing, fungal keratitis is still severe, refractory and vision-threatening disease.

Contact Lens-Related Infectious Keratitis with White Plaque Formation Caused by Corynebacterium propinquum.

We report the first case of Corynebacterium propinquum keratitis in the compromised cornea of a diabetic patient wearing therapeutic contact lenses. The strain was identified to the species level based on sequencing of the 16S rRNA gene and RNA polymerase ß-subunit-encoding gene (rpoB). Ophthalmologists should be aware of nondiphtherial corynebacterial infection of compromised corneas.

Development of an immunochromatographic assay kit using fluorescent silica nanoparticles for rapid diagnosis of Acanthamoeba keratitis.

We developed an immunochromatographic assay kit that uses fluorescent silica nanoparticles bound to anti-Acanthamoeba antibodies (fluorescent immunochromatographic assay [FICGA]) and evaluated its efficacy for the detection of Acanthamoeba and diagnosis of Acanthamoeba keratitis (AK). The sensitivity of the FICGA kit was evaluated using samples of Acanthamoeba trophozoites and cysts diluted to various concentrations. A conventional immunochromatographic assay kit with latex labels (LICGA) was also evaluated to determine its sensitivity in detecting Acanthamoeba trophozoites. To check for cross-reactivity, the FICGA was performed by using samples of other common causative pathogens of infectious keratitis, such as Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans. Corneal scrapings from patients with suspected AK were tested with the FICGA kit to detect the presence of Acanthamoeba, and the results were compared with those of real-time PCR. The FICGA kit detected organisms at concentrations as low as 5 trophozoites or 40 cysts per sample. There were no cross-reactivities with other pathogens. The FICGA was approximately 20 times more sensitive than the LICGA for the detection of Acanthamoeba trophozoites. The FICGA kit yielded positive results for all 10 patients, which corresponded well with the real-time PCR results. The FICGA kit demonstrated high sensitivity for the detection of Acanthamoeba and may be useful for the diagnosis of AK.

Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs.

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.

Severe fungal sclerokeratitis caused by Metarhizium anisopliae: a case report and literature review.

To date, there has been only one published report on the infectious sclerokeratitis caused by Metarhizium anisopliae, which is an entomopathogenic fungus. Regarding corneal infection, three reports have been published to date. Although the prognoses of the corneal infections are favourable, prognosis when scleral infection is involved is very poor. A 76-year-old patient presented with foreign body sensation in the left eye. Microscopic examination with Fungi Flora Y staining of the corneal scraping revealed fungal infection. The conjunctiva was melted by the infection over a wide area. Although intensive medications were administered, an emergency surgery was necessary because scleral thinning, corneal perforation and lens prolapse occurred. The fungal isolate was identified as M. anisopliae by sequencing the internal transcribed spacer region. Herein, we report the second known case worldwide of M. anisopliae sclerokeratitis, and we review the literature related to the ocular infections.