Ocular Samples Research Articles

Age-Related Changes to Human Tear Composition

We characterize age-associated alterations in the expression of inflammatory mediators and tissue remodeling factors in human tears. A total of 75 consecutive volunteers (32 male/44 female; 19-93 years) underwent clinical assessment of ocular surface status, ocular surface disease index (OSDI) grading and tear sampling. The volunteers were categorized into three groups: young (18-40 years), middle-aged (41-60 years), and old (>60 years). Total protein profiles and chip-based protein array evaluations were conducted to investigate the expression of 60 potential candidates, including pro-/anti-inflammatory mediators and tissue remodeling factors. Appropriate validations were performed using conventional assays. Multiple comparisons for regression between potential candidates and age were performed, as well as statistical analyses among the three age groups. Nonpooled samples were used for quantifications. Pearson analysis of chip-arrays identified 9 of 60 potential candidates. Specifically, IL-8, IL-6, and regulated on activation, normal T cell expressed and secreted (RANTES; P < 0.0083) protein as well as matrix metalloproteinase (MMP)-1, IL-3, and TNF-α (P < 0.05) correlated positively with aging. MIP-3β showed an opposite tendency. Western blot and ELISA analysis corroborated the array data. OSDI grading did not correlate with aging. Dynamic changes to tear protein profiles occur with aging. Our study identifies the expression of IL-8, IL-6, RANTES, MMP-1, and MIP-3β as increasing with age. These select inflammatory and matrix remodeling factors may be relevant to the development of novel diagnostic tools and therapeutics in the context of age-related ocular surface disease.

Immunogenicity testing of therapeutic antibodies in ocular fluids after intravitreal injection.

High drug concentrations in ocular fluids after intravitreal administration preclude the use of drug-sensitive immunoassays. A drug-tolerant immunoassay is therefore desirable for immunogenicity testing in ophthalmology. Immune complex (IC) antidrug antibody (ADA) assays were established for two species. The assays were compared with the bridging assay in ocular and plasma samples from two preclinical studies. The IC assays showed high drug tolerance, which enabled a reliable ADA detection in ocular fluids after intravitreal administration. The IC assays were superior to the bridging assay in the analysis of ocular fluids with high drug concentrations. The IC assay allows a reliable ADA detection in matrices with high drug concentrations, such as ocular fluids.

Development and application of two multiplex real-time PCR assays for detection and speciation of bacterial pathogens in the koala.

Infectious diseases have contributed to the decline in the health of koala ( Phascolarctos cinereus) populations in the wild in some regions of Australia. Herein we report the development and validation of 2 multiplex real-time PCR (rtPCR) panels for the simultaneous detection of Mycoplasma spp., Ureaplasma spp., Bordetella bronchiseptica, and Chlamydia, including speciation and quantification of Chlamydia, in ocular, reproductive, and nasal swab samples in addition to semen and male urogenital and reproductive tissues, from koalas. Each rtPCR panel was developed for use as a single-tube reaction using pathogen-specific primers and fluorescently labeled probe sets. DNA extracted from reference strains and isolates was used for validation of sequence gene targets for the multiplex rtPCR panels. Each panel was shown to be sensitive and specific in detecting and differentiating the bacterial pathogens. The multiplex rtPCR panels were used to screen clinical samples from free-ranging and hospitalized koalas for multiple pathogens simultaneously. The multiplex rtPCR will improve turnaround time compared to individual-pathogen rtPCR methods used, to date, for confirmation of diagnosis and will provide the wildlife clinician with the ability to make treatment decisions more rapidly.

Ebola Virus Persistence in Ocular Tissues and Fluids (EVICT) Study: Reverse Transcription-Polymerase Chain Reaction and Cataract Surgery Outcomes of Ebola Survivors in Sierra Leone

BackgroundEbola virus disease (EVD) survivors are at risk for uveitis during convalescence. Vision loss has been observed following uveitis due to cataracts. Since Ebola virus (EBOV) may persist in the ocular fluid of EVD survivors for an unknown duration, there are questions about the safety and feasibility of vision restorative cataract surgery in EVD survivors. MethodsWe conducted a cross-sectional study of EVD survivors anticipating cataract surgery and patients with active uveitis to evaluate EBOV RNA persistence in ocular fluid, as well as vision outcomes post cataract surgery. Patients with aqueous humor that tested negative for EBOV RNA were eligible to proceed with manual small incision cataract surgery (MSICS). FindingsWe screened 137 EVD survivors from June 2016 – August 2017 for enrolment. We enrolled 50 EVD survivors; 46 with visually significant cataract, 1 with a subluxated lens, 2 with active uveitis and 1 with a blind painful eye due to uveitis. The median age was 24.0years (IQR 17–35) and 35 patients (70%) were female. The median logMAR visual acuity (VA) was 3.0 (Snellen VA Hand motions; Interquartile Range, IQR: 1.2-3.0, Snellen VA 20/320 – Hand motions). All patients tested negative for EBOV RNA by RT-PCR in aqueous humor/vitreous fluid and conjunctiva at a median of 19months (IQR 18-20) from EVD diagnosis in Phase 1 of ocular fluid sampling and 34months (IQR 32-36) from EVD diagnosis in Phase 2 of ocular fluid sampling. Thirty-four patients underwent MSICS, with a preoperative median VA improvement from hand motions to 20/30 at three-month postoperative follow-up (P<0.001). InterpretationEBOV persistence by RT-PCR was not identified in ocular fluid or conjunctivae of fifty EVD survivors with ocular disease. Cataract surgery can be performed safely with vision restorative outcomes in patients who test negative for EBOV RNA in ocular fluid specimens. These findings impact the thousands of West African EVD survivors at-risk for ocular complications who may also require eye surgery during EVD convalescence.

Melanin Bleaching With Warm Hydrogen Peroxide and Integrated Immunohistochemical Analysis: An Automated Platform

Diagnosing melanocytic lesions is among the most challenging problems in the practice of pathology. The difficulty of physically masking melanin pigment and the similarity of its color to commonly used chromogens often complicate examination of the cytomorphology and immunohistochemical staining results for tumor cells. Melanin bleach can be very helpful for histopathological diagnosis of heavily pigmented melanocytic lesions. Although various depigmentation methods have been reported, no standardized methods have been developed. This study developed a fully automated platform that incorporates hydrogen peroxide-based melanin depigmentation in an automated immunohistochemical analysis. The utility of the method was tested in 1 cell block of malignant melanoma cells in pleural effusion, 10 ocular melanoma tissue samples, and 10 cutaneous melanoma tissue samples. Our results demonstrated that the proposed method, which can be performed in only 3 hours, effectively preserves cell cytomorphology and immunoreactivity. The method is particularly effective for removing melanin pigment to facilitate histopathological examination of cytomorphology and for obtaining an unmasked tissue section for immunohistochemical analysis.

Evaluation of a Chlamydia trachomatis-specific, commercial, real-time PCR for use with ocular swabs

BackgroundTrachoma, the leading infectious cause of blindness worldwide, is caused by conjunctival Chlamydia trachomatis infection. Trachoma is diagnosed clinically by observation of conjunctival inflammation and/or scarring; however, there is evidence that monitoring C. trachomatis infection may be required for elimination programmes. There are many commercial and ‘in-house’ nucleic acid amplification tests for the detection of C. trachomatis DNA, but the majority have not been validated for use with ocular swabs. This study evaluated a commercial assay, the Fast-Track Vaginal swab kit, using conjunctival samples from trachoma-endemic areas. An objective, biostatistical-based method for binary classification of continuous PCR data was developed, to limit potential user-bias in diagnostic settings.MethodsThe Fast-Track Vaginal swab assay was run on 210 ocular swab samples from Guinea-Bissau and Tanzania. Fit of individual amplification curves to exponential or sigmoid models, derivative and second derivative of the curves and final fluorescence value were examined for utility in thresholding for determining positivity. The results from the Fast-Track Vaginal swab assay were evaluated against a commercial test (Amplicor CT/NG) and a non-commercial test (in-house droplet digital PCR), both of whose performance has previously been evaluated.ResultsSignificant evidence of exponential amplification (R2 > 0.99) and final fluorescence > 0.15 were combined for thresholding. This objective approach identified a population of positive samples, however there were a subset of samples that amplified towards the end of the cycling protocol (at or later than 35 cycles), which were less clearly defined. The Fast-Track Vaginal swab assay showed good sensitivity against the commercial (95.71) and non-commercial (97.18) tests. Specificity was lower against both (90.00 and 96.55, respectively).ConclusionsThis study defined a simple, automated protocol for binary classification of continuous, real-time qPCR data, for use in an end-point diagnostic test. This method identified a population of positive samples, however, as with manual thresholding, a subset of samples that amplified towards the end of the cycling program were less easily classified. When used with ocular swabs, the Fast-Track Vaginal swab assay had good sensitivity for C. trachomatis detection, but lower specificity than the commercial and non-commercial assays it was evaluated against, possibly leading to false positives.

In vitro activity of hybrid lavender essential oils against multidrug resistant strains of Pseudomonas aeruginosa.

Lavender is an evergreen shrub native to Northern Africa and other mountainous Mediterranean regions. It grows throughout Southern Europe, the United States, and Australia. Lavender essential oil has been used since ancient times and is known for its anti-inflammatory, antidepressant, antiseptic, antifungal and antimicrobial properties. in this study, the antimicrobial activity of two Lavender essential oils (Lavanda sumian and Lavanda grosso) against 16 multidrug-resistant P. aeruginosa strains from clinical ocular samples taken from migrant patients has been investigated. The in vitro cytotoxic activity on human Wong-Kilbourne derivative (WKD) conjunctiva cells from healthy patients and nitric oxide synthase (NOS) activity on murine macrophage (J774.1A) were also evaluated. L. sumian showed lower antimicrobial activity when compared to L. grosso. Both lavender oils tested had no cytotoxic effect at very low concentrations, mostly L. grosso. The essential oils extracted from L. sumian and L. grosso significantly reduced NOS in a cell model. Increase in drug resistance and lack of new antibiotics may encourage the development of natural antimicrobial treatments.

Endogenous endophthalmitis secondary to melioidosis in paediatric patients: Case series and review article

Endogenous endophthalmitis is a devastating infection of the eye which can lead to permanent blindness. We report two rare cases of paediatric endogenous endophthalmitis secondary to melioidosis with contrasting visual outcomes. Both patients presented with acute painful visual loss with poor vision on presentation after exposure to open water sources (swimming at two separate locations with waterfalls). Both were diagnosed to have melioidosis endogenous endophthalmitis based on the ocular features clinically and via positive melioidosis serology. They otherwise did not exhibit any symptoms and signs suggestive of systemic melioidosis infection. Even though the two children demonstrated severe ocular involvement, ocular culture from vitreous and aqueous sampling taken from one of the patients did not yield any positive results. No ocular sampling was taken from the other patient. After standard antimicrobial treatment, the first patient responded well with good visual recovery without requiring any surgical intervention for the endophthalmitis. In contrast, our second patient ended up with poor visual outcome despite undergoing multiple intravitreal antibiotic injections and early pars plana vitrectomy. This is because he developed extensive retinal detachment due to the aggressive ocular infection. The diagnosis of endogenous endophthalmitis due to ocular melioidosis remains challenging and requires a high index of suspicion in areas endemic for the causative organism. Early empirical antibiotic treatment should be initiated in suspicious cases, even though the treatment outcomes may vary greatly.

Serological and Molecular Identification of Infectious Bovine Rhinotrachetitis Virus Isolation and Adaptation in Embryonated Chicken Eggs

Infectious bovine rhinotracheitis virus (IBRV) is a major pathogen in livestock animals and had led to significant economic losses to the industrial production worldwide. However, IBR symptoms are not life-threatening. The present study was achieved to make seroprevalence and isolation of bovine herpes virus type (BHV-1) from nasal and ocular swabs samples of suspected cows and buffaloes, identification by serological and polymerase chain reaction (PCR). Total of 380 blood samples were collected from suspected 287 cattle and 93 buffaloes in different districts at Beni-suef and El-Fayoum governorates in winter 2017. Nasal and ocular discharge swabs were collected from cattle and buffaloes with clinical respiratory signs (Nasal discharge, cough, lachrymal discharge with or without mild diarrhea and higher body temperature). A total of 106 (27.89%) samples were positive by indirect ELISA and the positivity of 80 (27.87%) samples from cattle and 26 (27.96%) samples from buffaloes located at different centers at Beni-Suif and El-Fayoum governorates. Virus was isolated from nasal and ocular discharges swabs samples and it was adapted in chorioallantoic membrane (CAM) of 11-day-old embryonated chickens eggs and in MDBK cells. Virus-infected CAM showed congestion, edematous vacuole, thickening small foci ranged from 2 to 3 mm in diameter, scattered on CAM membrane and MDBK cell line inoculated blind serial passages at 3rd passage showed characteristic of cytopathic effect (CPE). Identification of virus isolated on CAM and infected cell culture fluid gave precipitation against positive specific anti-BHV-1 immune serum by AGPT and clear blue zone by Dot ELISA, absent of pock lesion in pock reduction test (PRT), and confirmed by PCR with product size of 175 bp. Finally BHV-1 virus was isolated from nasal and ocular discharges of cattle and buffaloes but the further extensive study still need for clear final classification by phylogenic analysis.

Direct genetic characterization of Toxoplasma gondii from clinical samples from Denmark: not only genotypes II and III.

Genetic variation within Toxoplasma gondii can have both clinical and epidemiological significance, while the genotypes circulating in many parts of the world, including the Nordic country Denmark, are still unknown. We genetically characterized T. gondii strains that had been detected in human clinical samples in Denmark in 2011-2016. Samples that had tested positive for T. gondii DNA and had a quantification cycle value <33 were included in this study and subjected to direct genetic characterization of T. gondii based on length-polymorphism of 15 microsatellite markers. A total of 23 DNA samples from 22 individual patients were analyzed. The results were consistent with genotype II with 15/15 markers amplified from seven samples from the central nervous system (CNS) including two samples from one patient, four ocular samples, and one unspecified sample; with genotype III with 15/15 markers amplified from two ocular samples; with genotype Africa 1 with 15/15 markers amplified from one amniotic fluid sample and from one CNS-sample; with atypical genotype with 15/15 markers amplified from one CNS-sample and with 11/15 markers amplified from one CNS-sample; and with HG12-like genotype with 9/15 markers amplified from one CNS-sample. Genotype II, which is endemic in Europe, was predominant, but more than a third of the successfully genotyped strains were non-type-II. The possibility that clinical toxoplasmosis is caused by a strain that is not considered endemic to the region is definitely not negligible.

Does azithromycin given to women in labour decrease ocular bacterial infection in neonates? A double-blind, randomized trial

BackgroundVertical transmission can result in neonatal infection and disease. Reducing the transmission of bacterial pathogens from mother to infant may be an effective means of preventing neonatal infection, including bacterial conjunctivitis.MethodsIn a double-blind, randomized trial, we assessed the effect of administering a single dose of oral azithromycin to women in labour on bacterial colonization of the neonate. A reduction in purulent neonatal conjunctivitis was a secondary objective of the trial. Ocular samples were collected from the lower fornix of infants presenting with clinical signs of purulent conjunctivitis during the first eight weeks of life. Incidence of purulent conjunctivitis was compared between trial arms. Bacterial infection was assessed using PCR and incidence of purulent conjunctivitis due to bacteria was also compared between arms.ResultsForty of 843 infants (4.7%) presented clinical signs of purulent conjunctivitis. No significant difference in incidence of purulent conjunctivitis was seen between azithromycin and placebo arms [4.3% (18/419) versus 5.2% (22/424), OR = 0.82, 95% CI (0.44,1.54), p = 0.628]. S. aureus was the most commonly identified pathogen, detected in 38% of cases. Incidence of purulent-conjunctivitis due to bacterial infection was lower in the azithromycin arm [1.2% (5/419) versus 3.8% (16/424), OR = 0.31, 95% CI (0.12–0.82), p = 0.025)]. The incidence of gram-positive bacteria was also lower in the azithromycin arm [1.0% (4/419) versus 3.3% (14/424), OR = 0.28, 95%CI (0.10–0.82), p = 0.029].ConclusionsOral azithromycin given to women during labour may have the potential to reduce the incidence of bacterial neonatal conjunctivitis.Trial registrationClinicalTrials.gov, identifier NCT01800942, registration date 26 Feb 2013.

Ocular Histopathologic Features of Congenital Zika Syndrome

Congenital Zika syndrome (CZS) is known to be associated with severe malformations in newborns. Although microcephaly is the hallmark of this disease, the ocular findings are important given the severe visual impairment that has been observed in these patients. Regardless of the increased number of CZS cases reported, to date, no studies have described the ocular histopathologic findings of this entity. To evaluate the presence of Zika virus (ZIKV) antigens and describe the associated ocular histopathologic features of 4 cases of CZS. In this observational case series performed from June 19, 2015, through April 30, 2017, ocular tissue samples from 4 deceased fetuses with a diagnosis of CZS from the National Institute of Health in Colombia were sent to the Florida Lions Ocular Pathology Laboratory for evaluation. The microscopic features of each specimen were described, and immunostaining was performed using a ZIKV NS2B protein antibody. Ocular tissue samples from the 4 deceased fetuses (2 female, 2 male) ranging from 21.5 to 29 weeks' gestation with a diagnosis of CZS were studied. The 4 eyes manifested with pupillary membranes, immature anterior chamber angles, loss of pigment and thinning of the retinal pigment epithelium, choroidal thinning, undifferentiated nuclear layers of the retina, and a perivascular inflammatory infiltrate within the choroid. The optic nerve, present in 2 of the eyes, demonstrated atrophy. Expression of ZIKV antigen was present in the iris in cases 1, 3, and 4; the neural retina and choroid in case 1; and in the optic nerve in case 4. Loss of retinal pigment epithelium, the presence of a thin choroid, a perivascular choroidal inflammatory infiltrate, and atrophic changes within the optic nerve were consistently present. These findings may be attributed to ZIKV infection and warrant further study.

Recurrent herpetic keratitis despite antiviral prophylaxis: A virological and pharmacological study

Recurrent herpes simplex keratitis (HSK) is a leading infectious cause of blindness in industrialized countries. Antiviral prophylaxis (AVP) may fail to prevent recurrence of HSK due to viral resistance, inadequate dosing, or poor patient compliance. In this prospective multicenter study, we enrolled immunocompetent patients with recurrent HSK despite AVP. Ocular samples were tested by PCR for herpes simplex virus 1 (HSV-1). HSV-1 drug resistance was assessed with a genotypic assay based on UL23 and UL30 gene sequencing. After curative full dose valacyclovir (VACV) treatment was started, peak and trough acyclovir (ACV) plasma concentrations were measured, and patient compliance to AVP was assessed with a questionnaire.The study sample was comprised of 43 patients. Six (14%) patients were positive for HSV-1 using PCR, of whom 5 (83%) harbored genotypically ACV-resistant (ACVR) virus, due to mutations in UL23 (n = 4) or UL30 (n = 1). Disease duration was statistically significantly longer in patients with viral resistance compared to other HSK patients [35.5 ± 23.4 years (range, 6.8–68.4 years) versus 11.1 ± 12.3 years (range, 0.8–56.3 year) respectively; Mann-Whitney p = 0.01)].While patients were treated with full dose VACV, trough ACV plasma concentrations were below the threshold for ACV sensitivity in 9.5% of cases, and compliance was poor in 5.3% of cases.To summarize, HSV-1 resistance to ACV seems to be a significant cause of failure of prophylaxis in patients with HSK and is associated with longer disease duration. Most PCR-positive samples contained genotypically ACVR virus and identification may aid in adapting treatment. Incomplete 24-h drug coverage may also explain some cases of failure of prophylaxis.

Diagnostic Approach to Ocular Infections Using Various Techniques From Conventional Culture to Next-Generation Sequencing Analysis.

Ocular infection is caused by both endogenous (resident) and exogenous (environmental) microbes. As the ocular surface interacts with both outer environment and its own resident microbiota, clinical ocular samples are predicted to contain a diverse set of microorganisms. Microscopy of sample smears is an important step in the diagnostic process of infectious diseases to interpret the culture results. Traditional culture techniques have several limitations in the detection and/or identification of uncharacterized bacteria of environmental origin. Molecular biological techniques, such as polymerase chain reaction of pathogen-specific virulence genes, 16S rRNA gene clone library analysis, and next-generation sequencing of 16S rDNA amplicons, compensate for diagnostic culture techniques in diagnosing infectious diseases. These techniques are expected to provide novel insights into the ocular microbiota and pathology of ocular infections. In this article, we describe various ocular infections, including contact lens-related keratitis, silicone buckle infection, and dacryocystitis, which were analyzed using molecular biological techniques. The advantages and disadvantages of these highly sensitive and inclusive microbiological detection systems for ocular infections are discussed.

Bacterial pathogens associated with external ocular infections alongside eminent proportion of multidrug resistant isolates at the University of Gondar Hospital, northwest Ethiopia

BackgroundExternal ocular infection is a public health problem in Ethiopia. The aim of this study was to determine the prevalence and antimicrobial susceptibility pattern of external ocular bacterial infections.MethodsA cross sectional study was conducted at the University of Gondar Hospital among patients seeking health services at the Department of Ophthalmology from January to April, 2016. All patients with suspected external ocular infections were examined under slit lamp microscope. External ocular samples were collected using aseptic techniques. All samples were investigated by culture and bacteria were identified using standard methods. Drug susceptibility test was done using the Kirby-Bauer Disk diffusion method according to the guidelines of Clinical and Laboratory Standard Institute (CLSI).ResultA total of 312 samples were collected and 58.3% were culture positive. The proportion of Gram positive bacterial pathogens was (88%), and Staphylococcus aureus (50.3%) was the predominantly isolated pathogen, followed by Coagulase negative staphylococci (CoNS) (33.5%) and Klebsiella species (4.7%). Conjunctivitis was the dominant clinical feature, but a high positive result for bacterial pathogens was observed among patients with dacryocystitis cases. The Gram positive bacterial isolates were susceptible to ciprofloxacin, chloramphinicol, amoxicillin-clavulanate and ceftriaxone. However, 65% of these Gram positive bacterial pathogens showed resistance to penicillin, ampicillin and amoxicillin. The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infection was 24% and multidrug resistance (MDR) was observed in 87% of the isolated bacteria.ConclusionConjunctivitis was the dominant ophthalmic disease followed by blepharitis. The dominant bacteria species was S. aureus and MRSA infection is increasingly prevalent. The overall MDR bacterial pathogen proportion was very high. The high prevalence of MRSA and MDR bacterial pathogens dictate the need for effective prevention as important as for therapies.

Epidemiological and molecular characteristics of Chlamydia psittaci from 8 human cases of psittacosis and 4 related birds in Argentina

In Argentina, the epidemiological and molecular characteristics of Chlamydia psittaci infections are still not sufficiently known. A total of 846 respiratory and 10 ocular samples from patients with suspected human psittacosis were tested for C. psittaci from January 2010 to March 2015. Four samples of birds related to these patients were also studied. Forty-eight samples were positive for C. psittaci by a nested PCR. The molecular characterization of twelve C. psittaci PCR-positive samples received in the National Reference Laboratory INEI-ANLIS “Dr. Carlos G. Malbrán”, Buenos Aires, Argentina was performed. Eight positive samples from humans and four from birds were genotyped by ompA gene sequencing. C. psittaci genotype A was found in all human samples and in the related birds. This report contributes to our increasing knowledge of the epidemiological and molecular characteristics of C. psittaci to conduct effective surveillance of its zoonotic infections.

Pythium Insidiosum Keratitis: Histopathology and Rapid Novel Diagnostic Staining Technique.

To elucidate the histopathology of Pythium insidiosum keratitis and to describe a novel, simple, and rapid staining technique for identification of oomycete Pythium insidiosum and to differentiate it from fungi. This is a laboratory investigation study of 38 nonconsecutive cases (37 ocular samples and 1 colonic biopsy); 14 microbiologically diagnosed as Pythium insidiosum keratitis and 24 as fungal keratitis. Review of clinical, demographic details, microbiological results, and identification of cases that necessitated evisceration was performed. Reevaluation of histopathology slides was done using stains such as hematoxylin-eosin, Gomori methenamine silver (GMS), periodic acid-Schiff (PAS), potassium iodide-sulfuric acid (IKI-H2SO4). Morphology, degree, and nature of inflammation and load, distribution, and staining results of Pythium insidiosum and its comparison with fungi were studied. Delay in zoospore formation, failure of growth, and delay in identification of Pythium were the main cause of evisceration. Corneal pythiosis showed epithelial ulceration, stromal destruction, and varying inflammation; load and distribution of Pythium were inversely proportional to inflammation. The filaments were commonly wide, with admixed narrower structures and uncommonly involved Descemet membrane. The oomycete was not discretely discerned with PAS stain and stained distinctly with GMS stain and IKI-H2SO4 stain (100% sensitive). In comparison, fungal organisms stained well with PAS and GMS stain, but not with IKI-H2SO4 stain (100% specific). Pythium insidiosum keratitis is perhaps not more devastating than fungal keratitis but late diagnosis, misdiagnosis, and treatment as fungal infection are major heralds. Early diagnosis may markedly improve the patient outcome. IKI-H2SO4 is a cost-effective, simple, sensitive, and specific stain for the diagnosis of oomycete Pythium.

MOLECULAR DETECTION OF INFECTIOUS PATHOGENS OF THE UPPER RESPIRATORY TRACT IN CAPTIVE NONDOMESTIC FELIDS.

Upper respiratory tract disease is a complex infectious disease process with multiple pathogens involved. Identification of infectious agents in wild animals is of great importance for wildlife conservation. The aim of this study was to evaluate the molecular detection of feline herpesvirus type 1, feline calicivirus (FCV), Bordetella bronchiseptica , Chlamydophila felis , and Mycoplasma felis using ocular and nasal swabs in three species of captive nondomestic felids. Mycoplasma felis was detected in two ocular samples of Puma concolor and in one nasal sample of one Panthera onca . FCV was detected in association with M. felis in one P. concolor . The other pathogens tested were not detected. To the authors' knowledge, this is the first report of M. felis in nondomestic felids from Brazil.

Retrospective report of antimicrobial susceptibility observed in bacterial pathogens isolated from ocular samples at Mount Sinai Hospital, 2010 to 2015

BackgroundAntimicrobial resistance has emerged as a major threat to global public health. Thus, the surveillance of changes in antimicrobial resistance in local and global settings is a paramount necessity. While many studies have tracked antimicrobial resistance, only a small percentage surveyed ocular isolates. The purpose of this study was to report the in vitro susceptibility of bacterial pathogens isolated from ocular samples in New York, NY from 2010 to 2015.MethodsA retrospective review of ocular isolates was conducted. All organisms were collected by 25 separate inpatient wards and outpatient clinics, and were analyzed by the clinical microbiology laboratory at Mount Sinai Hospital. Clinical Laboratory and Standards Institute (CLSI) guidelines were followed for susceptibility testing and breakpoint interpretations.ResultsA total of 549 bacterial organisms were isolated from 1664 cultures (33%) during the 6-year study period. Of these, 358 isolates (65.2%) underwent susceptibility testing. 182 (50.8%) isolates were Gram-positive. The most common Gram-positive bacterium was Staphylococcus aureus (62.1%). Methicillin-resistance decreased in S. aureus isolates (31.3% in 2010, 14.1% in 2015) but was without significant change (p = 0.25). When analyzing all S. aureus isolates recovered during the study period, there were significantly more methicillin-resistant S. aureus (MRSA) isolates resistant to fluoroquinolones (p <0.0001), erythromycin (p <0.0001), and trimethoprim/sulfamethoxazole (TMP/SMZ; p <0.05). Overall, Streptococcus pneumoniae isolates showed reduced susceptibility to erythromycin, but were otherwise susceptible to the other antimicrobials tested. Haemophilus influenzae (26.1%) and Pseudomonas aeruginosa (23.9%) were the most common Gram-negative bacteria isolated. Resistance to ampicillin and TMP/SMZ was observed in several of the H. influenzae isolates. P. aeruginosa isolates did not show high resistance overall, however, it was noted that isolates resistant to meropenem were also resistant to other antimicrobials (p < 0.01).ConclusionOverall, antimicrobial resistance was infrequent for the Gram-negative and Gram-positive bacteria analyzed. While the MRSA isolates demonstrated increased resistance to multiple antimicrobial classes, this is expected for this pathogen. Due to the continued use of broad-spectrum oral and systemic antimicrobials to treat ocular infections, findings of this study and other surveillance studies specific to ocular isolates should be used as resources in effective decision making in the treatment of ocular disease.

Establishment of Multiplex Solid-Phase Strip PCR Test for Detection of 24 Ocular Infectious Disease Pathogens.

To establish and evaluate a new multiplex solid-phase strip polymerase chain reaction (strip PCR) for concurrent detection of common ocular infectious disease pathogens. A new multiplex strip PCR was established to detect 24 common ocular infectious disease pathogens: herpes simplex virus (HSV) 1, HSV2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus (HHV) 6, HHV7, HHV8, human T-cell lymphotropic virus (HTLV)-1, adenovirus, Mycobacterium tuberculosis, Treponema pallidum, Propionibacterium acnes (P. acnes), bacterial 16S ribosomal RNA (rRNA), Candida species (Candida sp.), C. glabrata, C. krusei, Aspergillus, Fusarium, fungal 28S rRNA, Toxoplasma (T. gondii), Toxocara, Chlamydia trachomatis (C. trachomatis), and Acanthamoeba. Strip PCR was tested with a negative control (distilled water) and standard positive control DNA. Cutoffs of quantification cycle (Cq) values were determined with noninfectious ocular samples to avoid false-positives caused by contamination with P. acnes, bacterial 16S, and fungal 28S from reagents and ocular surfaces. A pilot study to evaluate the strip PCR was performed using infectious ocular samples (aqueous humor, vitreous, cornea, and tears) by strip PCR and previously developed capillary-type multiplex PCR and quantitative real-time PCR (qPCR). Strip PCR was verified with negative and positive controls. Strip PCR rapidly detected HSV1, HSV2, VZV, EBV, CMV, HHV6, HHV7, HTLV-1, adenovirus, P. acnes, bacterial 16S, Candida sp., C. glabrata, Aspergillus, fungal 28S, T. gondii, C. trachomatis, and Acanthamoeba in patient samples. The sensitivity was comparable to that of qPCR. Our novel strip PCR assay is a simple, rapid, and high-sensitivity method for detecting ocular infectious disease pathogens.