Use of different stains for microscopic evaluation for the diagnosis of Pythium keratitis

PurposeTo evaluate the efficacy of various staining techniques for detection of Pythium in keratitis cases.MethodsData of nineteen consecutive culture-positive cases of Pythium keratitis were retrospectively analysed. Corneal scrapings and corneal buttons (in the cases which underwent therapeutic penetrating keratoplasty [TPK]) were sent for microbiological and histopathological examination. The direct smears were stained with Potassium hydroxide and calcofluor white (KOH ?+ ?CFW), Gram and Iodine–Potassium Iodide–Sulphuric Acid (IKI–H₂SO₄) stains. The corneal buttons were stained with Gomori's Methanamine Silver (GMS), Periodic Acid-Schiff (PAS) and Iodine–Potassium Iodide–Sulphuric Acid (IKI–H₂SO₄) stains. The positivity of various stains in detecting Pythium was studied.ResultsGram and KOH ?+ ?CFW staining from smear was done in 16 out of 19 (84.2%) cases. KOH ?+ ?CFW and Gram stains were suggestive of Pythium in 10 (62.5%) and 7 (43.8%) cases, respectively. IKI–H₂SO₄ staining in scraping samples was positive for Pythium in all the 4 (100%) cases in which it was performed. Half corneal buttons were positive for Pythium with IKI–H₂SO₄ stain as well as GMS stain in all the 18 cases that underwent TPK (100%). PAS stain showed weak to faint pink staining of Pythium filaments in 7 out of 18 cases (38.9%).ConclusionIKI–H₂SO₄ stain followed by KOH ?+ ?CFW stain detects Pythium filaments most accurately in corneal scraping samples from keratitis patients, although the differences were not statistically significant. The positivity of the stains depends on astute observation by an experienced ocular microbiologist and pathologist.     

Acanthamoeba keratitis in a mouse model using a novel approach

ContextAcanthamoeba is increasingly implicated in causing keratitis in patients wearing contact lens or ocular trauma and has a poor prognosis. Establishment of an animal model is critical to study the disease pathology, pathogenesis and to evaluate anti-amoebic drugs. Some studies have used contact lenses to establish Acanthamoeba keratitis (AK) in a mouse model, which is expensive and not very successful as lenses get dislodged.ObjectiveTo assess the feasibility of using parafilm (Bemis Company Inc., USA) as an alternative to contact lens for the establishment of AK in the mouse model.MethodsThirty-six Balb/c mice in three groups of six mice each for two strains of Acanthamoeba were used to induce AK. Three experimental approaches used were; i) Acanthamoeba impregnated contact lens, ii) Acanthamoeba impregnated parafilm and iii) scratching followed by inoculation of Acanthamoeba suspension. In all three models, tarsorrhaphy was performed. Infection was evaluated by clinical examination and also through microscopic examination of corneal scrapings and corneal sections.ResultsAK model was successfully established with parafilm whereas only one mouse developed AK with the use of contact lens and none with scratching and Acanthamoeba inoculation.ConclusionThe use of parafilm is convenient, reliable and cheaper and can be considered an alternative to contact lenses to induce AK in a mouse model.     

Fungal keratitis caused by a rare pathogen,Colletotrichum gloeosporioides,in an east coast city of China

BackgroundTo report a case of fungal keratitis caused by Colletotrichum gloeosporioides in an east coast city of China, which are rare pathogens that cause fungal keratitis in humans.MethodsA 52-year-old man whose right eye was injured by a branch of an apple tree during farm work was referred to our Hospital. He was examined by Slit-lamp and the HRT II-RCM confocal scanning microscope, thus suggesting filamentous. Orneal scrapings were acquired and then inoculated into Sabouraud medium incubated at 28 °C and 37 °C. In vitro antifungal susceptibility tests were performed following the CLSI M38-A2 for Filamentous Fungi. Surgical intervention was advised because the abscess in the anterior chamber of the right eye was not completely absorbed.ResultsThe Colletotrichum gloeosporioides isolate was identified by MALDI-TOF mass spectrometry and the BLAST after DNA sequencing of the amplified internal transcribed spacer (ITS) in rRNA. The patient's eye condition is under control and the patient's vision remains at the level of light perception (LP).ConclusionsWe report the rare keratitis caused by C. gloeosporioides in eastern China, which has not been published. Suddenly ocular trauma and old surgical intervention may be the risk factors associated with Colletotrichum keratitis.     

Reduced egg counts in mixed infections with Oestrus ovis and Haemonchus contortus : influence of eosinophils?

In all, 2 groups of lambs were infected either with Oestrus ovis first-instar larvae or with 10,000 third-stage larvae (L3) of Haemonchus contortus. Another group of lambs was infected with both parasites. Fecal nematode egg counts, plasma pepsinogen concentrations, specific O. ovis enzyme-linked immunosorbent assay (ELISA) antibodies, and blood eosinophil counts were monitored and compared to with the values recorded for a control group of uninfected lambs. There was no significant difference between the burden of H. contortus found in mixed and single infections. However, the nematode egg production was significantly depressed in mixed infections. O. ovis affects the population of H. contortus at least by decreasing the parasite egg output. This effect may be mediated through the increase in eosinophil production stimulated by the presence of O. ovis.     

Application of immunoperoxidase staining in the cytodiagnosis of herpes simplex keratitis

In corneal scraping smears from 13 patients with clinically suspected herpes simplex keratitis (HSK), HSK is demonstrated by means of peroxidase-antiperoxidase (PAP) technique with antisera to herpes simplex virus (HSV) in Papanicolaou-destained cellular samples. The staining for HSV antigen was present in seven cases of corneal scraping smears with superficial keratitis (dendritic and geographic ulcers) while six cases of stromal keratitis (deep keratitis) failed to show HSV antigen except in one case. Specific antigen for HSV was predominantly present in the cytoplasm rather than in the nucleus. Immunoreactions were negative with HSV antisera in patients with other infections and in those in a normal control group. Using the PAP technique, detection of HSV antigen in corneal scraping smears was of great value in the diagnosis of HSK, especially in cases of superficial keratitis.     

First case of Tropicoporus tropicalis keratitis in an immunocompetent host from India and review of the literature

Tropicoporus tropicalis is an environmental basidiomycete that has been implicated in nine cases of cutaneous (n = 7) and pulmonary (n = 2) human infections predominantly in chronic granulomatous disease patients. We report here the first case of keratitis caused by Tropicoporus tropicalis in a 40-year-old immunocompetent patient, who presented with sudden diminution of vision in right eye. Corneal scrapings revealed hyaline, septate hyphae in microscopy and culture showed growth of white non-sporulating mycelial growth which was confirmed as Tropicoporus tropicalis by sequencing of ITS region of 28S rDNA. The patient was initiated on topical voriconazole along with natamycin, gatifloxacin and atropine drops. However, despite treatment, corneal ulcer perforated, for which penetrating keratoplasty was performed. Thereafter, he was prescribed amphotericin B (AMB) drops sixteen times a day and ketoconazole 200 mg twice a day with no recurrence reported over one year of follow up. The case represents the first case of infection by this fungus from India and also is the first case to be reported in an immunocompetent host.     

Analysis of the herpes simplex virus type 1 UL6 gene in patients with stromal keratitis

Recent work suggests that herpes simplex virus (HSV) stromal keratitis in the mouse is caused by autoreactive T lymphocytes triggered by a 16 amino acid region of the HSV UL6 protein (aa299-314), Science 279, 1344-1347). In the present study we sought to determine whether genetic variation of this presumed autoreactive UL6 epitope is responsible for different pathogenic patterns of human HSV keratitis. To accomplish this, we sequenced the HSV UL6 gene from ocular isolates of 10 patients with necrotizing stromal keratitis, 7 patients with recurrent epithelial keratitis, and 8 patients with other forms of HSV keratitis. The sequences obtained predicted identical UL6(299-314) epitopes for all 25 viral isolates. Furthermore, the upstream sequence of all isolates was free of insertions, deletions, and stop codons. We conclude that different pathogenic patterns of human HSV keratitis occur independent of genetic variation of the HSV UL6 (299-314) epitope.     

Comparison of Oestrus ovis Metabolic and Somatic Antigens for the Immunodiagnosis of the Zoonotic Myasis Oestrosis by Immunoenzymatic Probes

Control of zoonosis implies reduction of infected animal hosts, and the first measure consists of a suitable and accurate detection test. An experimental study for determining the most appropriate antigen (metabolic or somatic) to be used in the detection of the oestrosis (Oestrus ovis) zoonotic myasis by means of immunoenzymatic probes was carried out. A flock of 23 uninfected goats was maintained under field conditions to allow their infection in Sassari (Sardinia, Italy). Caprine were bled monthly and serum samples processed by means of an iELISA. After comparing these results to the chronobiology of O. ovis, we proved that the IgG humoral response against the metabolic antigens increased only during the period of real risk of infestation (when adults fly, from May to September), whereas the absorbances against the somatic products were positive from the beginning of the study (in January, prior to infection). We concluded that the excretory/secretory products are most useful andsuitable for the immunodiagnosis of oestrosis in goats, because a direct relation between the development of O. ovis and the IgG humoral response is possible, allowing a more accurate diagnostic.     

Rough Lipopolysaccharide of Brucella abortus RB51 as a Common Antigen for Serological Detection of B. ovis,B. canis,and B. abortus RB51 Exposure Using Indirect Enzyme Immunoassay and Fluorescence Polarization Assay

Abstract

Rough lipopolysaccharide (RLPS) antigens were prepared from cultures of Brucella abortus RB51, B. ovis, and B. canis. The preparations were standardized by weight and tested with sera from cattle immunized with B. abortus RB51, sheep infected with B. ovis, and dogs infected with B. canis. Populations of unexposed animals of each species were also tested. The tests used were the indirect enzyme immunoassay (IELISA) using RLPS and the fluorescence polarization assay (FPA) using RLPS core fractions, labeled with fluorescein isothiocyanate. The IELISA using B. abortus RB51 RLPS antigen resulted in sensitivity and specificity values of 94.8% and 97.3%, respectively, when testing bovine sera, 98.5% and 97.8% when testing ovine sera, and 95.8% and 100% when testing dog sera. The IELISA using B. ovis RLPS antigen gave sensitivity and specificity values of 80.5% and 91.7%, respectively with bovine sera, 98.9% and 93.8% with sheep sera, and 70.8% and 79.8% with dog sera. The IELISA using B. canis RLPS antigen resulted in sensitivity and specificity values of 97.0% and 97.4%, respectively, with bovine sera, 96.2% and 96.3% with sheep sera, and 95.8% and 98.8% with dog sera. Labeling RLPS core from B. ovis and B. canis with fluorescein was not successful. B. abortus RB51 core labeled with fluorescein resulted in sensitivity and specificity values of 93.5% and 99.8%, respectively, with bovine sera and 78.1% and 99.0% with sheep sera. It was not possible to test the dog sera in the FPA.     

Vaginocervical stimulation of Ewes induces the rapid formation of a new bond with an alien young without interfering with a previous bond

Ewes form a selective olfactory memory for their lambs after 2 hr of mother–young interaction following parturition. Mothers will subsequently reject any strange lamb at suckling. The present study investigated whether artificial vaginocervical stimulation (VCS) allows the formation of a selective bond with an unfamiliar lamb and whether it interferes with the maintenance of the bond formed with the familiar lamb. At 2 hr postpartum, mothers were separated from their familiar lamb after having formed a selective bond with it and were given 10 min of mechanical VCS. In the “VCS + lamb” group (n = 24) an unfamiliar lamb was left with the ewe for 2 hr whereas in the “VCS no lamb” group (n = 26) the mother was left alone for the same period of time. Ewes of the “no VCS” group (n = 14) did not receive any VCS. In the majority of animals of the “VCS + lamb” group (23/24) VCS induced a complete acceptance of the unfamiliar lamb without any disruption of the bond previously formed with the familiar lamb. VCS or 2 hr of separation did not disrupt the maintenance of the selective bond initially formed with the familiar lamb since all the ewes of the “VCS no lamb” and “no VCS” groups accepted it at suckling. © 2010 Wiley Periodicals, Inc. Dev Psychobiol 52: 537–544, 2010.     

Lacrimal Duct Occlusion Is Associated with Infectious Keratitis

Background: To explore the prevalence of lacrimal duct obstruction in patients with infectious keratitis, and the necessity of lacrimal duct dredge in the treatment of human infectious keratitis.Methodology/Principle Findings: The design is prospective, non-control case series. Thirty-one eyes from twenty-eight continuous patients with infectious keratitis were included in this study. The presence/absence of lacrimal duct obstruction was determined by the lacrimal duct irrigation test. The diagnosis of infectious keratitis was made based on clinical manifestations, cornea scraping microscopic examination and bacterial/fungus culture. Diagnosis of viral keratitis was set up based on the recurrent history, deep neovascularization and typical outlook of the cornea scar. The treatment of keratitis included drugs, eye drops or surgery, while treatment of chronic dacryocystitis was lacrimal duct dredging with supporting tube implantation surgery. In the thirty-one eyes with infectious keratitis, fifteen suffered from fungal keratitis (48%), two bacterial keratitis (6%), and fourteen viral keratitis (45%). Eleven eyes (35%) from ten patients with infectious keratitis also suffered from lacrimal duct obstruction. In those cases, six eyes also suffered from lower canalicular obstruction, three nasolacrimal duct obstruction and chronic dacryocystitis, one a combination of upper and lower canalicular obstruction, one upper canalicular obstruction. After local and systemic applications of anti-bacterial, anti-viral, anti-fungal and anti-inflammatory drugs, twenty-eight eyes (90%) recovered within three weeks, while the ulceration of three patients required the lacrimal duct dredging and supporting tube implantation surgery for the healing.Conclusions: Herein, we first report that the prevalence of infectious keratitis is closely correlated to the occurrence of lacrimal duct obstruction. When both confirmed, simultaneous treatment of keratitis and lacrimal duct obstruction promptly is required. Further evaluation of mechanism, prevention and control of the diseases are warranted.     

Anti-oncospheral antibodies in the serum of lambs experimentally infected with eitherTaenia ovis orTaenia hydatigena

Using a peroxidase micro-enzyme-linked-immunosorbent assay (ELISA) method anti-oncospheral antibodies were demonstrated in sera from four lambs after primary and challenge infections withTaenia ovis orT. hydatigena. Antibodies assayed using homologous oncospheral antigen (OA) reached a peak by 3–4 weeks after primary infection and at 1–3 weeks post-challenge infection, but waned to pre-infective, background levels by 8–12 weeks after each infection. Antibodies assayed against antigens in strobilar or cystic larval extracts persisted for long periods after the initial infections and exhibited different kinetics of response from those demonstrated against OA. These antibodies showed increased levels after challenge infection. Oncospheral antigens did not seem to be species specific although they appeared to elicit a stage-specific response. It is suggested that the anti-oncospheral antibody response could be associated with protective or functional antibody.     

Serine-like proteolytic enzymes correlated with differential pathogenicity in patients with acute Acanthamoeba keratitis

Acute ocular infection due to free-living amoebae of the genus Acanthamoeba is characterized by severe pain, loss of corneal transparency and, eventually, blindness. Proteolytic enzymes secreted by trophozoites of virulent Acanthamoeba strains have an essential role in the mechanisms of pathogenesis, including adhesion, invasion and destruction of the corneal stroma. In this study, we analysed the relationship between the extracellular proteases secreted by clinical isolates of Acanthamoeba and the clinical manifestations and severity of disease that they caused. Clinical isolates were obtained from patients who showed typical symptoms of Acanthamoeba keratitis. Trophozoites were cultivated axenically, and extracellular proteins were collected from cell culture supernatants. Secreted enzymes were partially characterized by gelatin and collagen zymography. Acanthamoeba trophozoites secreted proteases with different molecular masses, proteolysis rates and substrate specificities, mostly serine-like proteases. Different enzymatic patterns of collagenases were observed, varying between single and multiple collagenolytic activities. Low molecular weight serine proteases were secreted by trophozoites associated with worse clinical manifestations. Consequently, proteolytic enzymes of some Acanthamoeba trophozoites could be related to the degree of their virulence and clinical manifestations of disease in the human cornea.     

Relapsing poly(peri)chondritis associated with fibrosing alveolar disease and antibodies to pneumocytes type II and clara cells

Summary A 62-year-old man with histological confirmed relapsing polychondritis showed chondritis of ears and nose, arthritis, keratitis and a hemolytic anemia. The bronchoalveolar lavage, computed tomography of the thorax and pulmonary function tests disclosed findings compatible with fibrosing alveolar disease. IgG antibodies to alveolar pneumocytes type II and bronchiolar Clara cells were detected by indirect immunofluorescense of human lung tissue. To our knowledge this is the first report of fibrosing alveolar disease in relapsing polychondritis and detection of antibodies to human pneumocytes type II and Clara cells.     

遗传缺陷性小鼠角膜炎模型的超微病理观察

目的 观察不同时段遗传缺陷性小鼠角膜炎模型角膜组织的形态学变化.方法 应用ENU诱变技术建立小鼠角膜炎模型;用透射电镜观察在不同时期角膜感染部位的超微结构改变,并辅以光镜观察.结果 成功建立小鼠模型;在模型小鼠出生后的不同时段中,电镜下小鼠角膜基质内可见有细菌感染、炎症细胞浸润、成纤维细胞及新生毛细血管增生等炎症改变.结论 透射电镜和光镜研究手段能有效地在组织和超微结构水平研究角膜感染时的动态变化,为研究该疾病提供可靠的形态学依据.