Cutibacterium modestum sp. nov., isolated from meibum of human meibomian glands, and emended descriptions of Cutibacterium granulosum and Cutibacterium namnetense.

An anaerobic and aerotolerant bacterium, strain M12T, was isolated from the meibum of inflamed human meibomian glands. Cells of the strain was Gram-stain-positive, non-spore-forming and non-motile rods. Growth on trypticase soy agar plates supplemented with 5 % sheep blood was fastest at 30-37 °C under anaerobic conditions. The 16S rRNA gene sequence of the strain revealed that it belongs to the genus Cutibacterium with a 98.0 % similarity value to the closest species, Cutibacterium acnes. Genome analysis of the strain with type strains of the other Cutibacterium species resulted in digital DNA-DNA hybridization values of 32.3-22.3% and average nucleotide identity (OrthoANI) values of 86.7-73.6 %. Biochemical and physiological analyses using API rapid ID 32A and API Coryne kits revealed relatively low reactivity of the strain compared with C. acnes and Cutibacterium namnetense. The most abundant major cellular fatty acid was iso-C15 : 0. Fermentation end-products from glucose were propionate, lactate, succinate and acetate. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. Major menaquinones were MK-9(H4), MK-9(H2) and MK-9. The major peaks of the MALDI-TOF mass spectrometry spectrum were at 3493, 3712, 6986 and 7424 Da. The DNA G+C content was 59.9 mol%. Based on these findings, we propose a novel species, Cutibacterium modestum. The type strain of C. modestum is M12T (=JCM 33380T=DSM 109769T). On the basis of further genomic analysis, we also provide emended descriptions of Cutibacterium granulosum (Prévot 1938) Scholz and Kilian 2016 and Cutibacterium namnetense (Aubin et al. 2016) Nouioui et al. 2018.

The Antimicrobial Activity of Multipurpose Disinfecting Solutions in the Presence of Different Organic Soils.

OBJECTIVE: During use, contact lens disinfecting solutions are exposed to tears and clinical microbial isolates. The current study was designed to test the performance of several disinfecting solution in the presence of organic soils or clinical isolates. METHODS: Standard and clinical isolates were exposed to the disinfecting solutions in the presence or absence of different organic soils. The number of microbial cells killed during disinfection was established by growing cells after disinfection on agar plates. RESULTS: The disinfecting activity of the povidone-iodine or hydrogen peroxide solutions was not affected by the organic soils or clinical isolates. The presence of yeast organic soil did not affect the performance of the disinfecting solutions when tested with standard microbial strains, but the addition of a model tear organic soil significantly reduced the disinfecting activity of the solutions containing various combinations of polyhexamethylene biguanide, polyquaternium-1, alexidine, and myristamindopropyl dimethylamine especially when tested against the standard fungal strains (reducing the effectiveness by between 0.5-4 log10) or the clinical bacterial isolates (reducing the effectiveness by between 0.5-3.5 log10). One disinfecting solution that contained polyquaternium-1 and myristamindopropyl dimethylamine had very poor activity against the clinical bacterial isolates in the absence or presence of either organic soil. CONCLUSIONS: Povidone-iodine or hydrogen peroxide disinfecting solutions are not affected by organic soils and are very active against clinical bacterial isolates. Disinfecting solutions containing combinations of polyhexamethylene biguanide, polyquaternium-1, alexidine, and myristamindopropyl dimethylamine are affected by model tear organic soil and may have poor activity against clinical isolates.

Clinical Utility of Noninvasive Method to Measure Specific Gravity in the Pediatric Population.

BACKGROUND: Clinicians rely on any combination of signs and symptoms, clinical scores, or invasive procedures to assess the hydration status in children. Noninvasive tests to evaluate for dehydration in the pediatric population are appealing. OBJECTIVE: The objective of our study is to assess the utility of measuring specific gravity of tears compared to specific gravity of urine and the clinical assessment of dehydration. METHODS: We conducted a prospective cohort convenience sample study, in a pediatric emergency department at a tertiary care children's hospital. We approached parents/guardians of children aged 6 months to 4 years undergoing transurethral catheterization for evaluation of urinary tract infection for enrollment. We collected tears and urine for measurement of tear specific gravity (TSG) and urine specific gravity (USG), respectively. Treating physicians completed dehydration assessment forms to assess for hydration status. RESULTS: Among the 60 participants included, the mean TSG was 1.0183 (SD = 0.007); the mean USG was 1.0186 (SD = 0.0083). TSG and USG were positively correlated with each other (Pearson Correlation = 0.423, p = 0.001). Clinical dehydration scores ranged from 0 to 3, with 87% assigned a score of 0, by physician assessment. Mean number of episodes of vomiting and diarrhea in a 24-hour period were 2.2 (SD = 3.9) and 1.5 (SD = 3.2), respectively. Sixty-two percent of parents reported decreased oral intake. CONCLUSION: TSG measurements yielded similar results compared with USG. Further studies are needed to determine if TSG can be used as a noninvasive method of dehydration assessment in children.

Glycan involvement in the adhesion of Pseudomonas aeruginosa to tears.

The human eye is constantly bathed by tears, which protect the ocular surface via a variety of mechanisms. The O-linked glycans of tear mucins have long been considered to play a role in binding to pathogens and facilitating their removal in the tear flow. Other conjugated glycans in tears could similarly contribute to pathogen binding and removal but have received less attention. In the work presented here we assessed the contribution of glycan moieties, in particular the protein attached N-glycans, presented by the broad complement of tear proteins to the adhesion of the opportunistic pathogen Pseudomonas aeruginosa, a leading cause of microbial keratitis and ulceration of the cornea. Our adhesion assay involved immobilising the macromolecular components of tears into the wells of a polyvinyl difluoride (PVDF) microtitre filter plate and probing the binding of fluorescently labelled bacteria. Three P. aeruginosa strains were studied: a cytotoxic strain (6206) and an invasive strain (6294) from eye infections, and an invasive strain (320) from a urinary tract infection (UTI). The ocular isolates adhered two to three times more to human tears than to human saliva or porcine gastric mucin, suggesting ocular niche-specific adaptation. Support for the role of the N-glycans carried by human tear proteins in the binding and removal of P. aeruginosa from the eye was shown by: 1) pre-incubation of the bacteria with free component sugars, galactose, mannose, fucose and sialyl lactose (or combination thereof) inhibiting adhesion of all the P. aeruginosa strains to the immobilised tear proteins, with the greatest inhibition of binding of the ocular cytotoxic 6206 and least for the invasive 6294 strain; 2) pre-incubation of the bacteria with N-glycans released from the commercially available human milk lactoferrin, an abundant protein that carries N-linked glycans in tears, inhibiting the adhesion to tears of the ocular bacteria by up to 70%, which was significantly more binding inhibition than by the same amount of intact human lactoferrin or by the plant-derived N-glycans released from the rice recombinant lactoferrin; 3) pre-incubation of the bacteria with N-linked glycans released from human tear proteins inhibiting the adhesion of the ocular P. aeruginosa strains to immobilised tear proteins; 4) inhibition by the N-glycans from lactoferrin of the ability of an ocular strain of P. aeruginosa to invade corneal epithelial cells; 5) removal of terminal sialic acid and fucose moieties from the tear glycoproteins with α2-3,6,8 neuraminidase (sialidase) and α1-2,3,4 fucosidase resulting in a reduction in binding of the UTI P. aeruginosa isolate, but not the adhesion of the ocular cytotoxic (6206) or invasive (6294) isolates. Glycosidase activity was validated by mass spectrometry. In all cases, the magnitude of inhibition of bacterial adhesion by the N-glycans was consistently greater for the cytotoxic ocular strain than for the invasive ocular strain. Ocular P. aeruginosa isolates seems to exhibit different adhesion mechanism than previously known PAI and PAII lectin adhesion. The work may contribute towards the development of glycan-focused therapies to prevent P. aeruginosa infection of the eye.

Short communication: Iodine concentrations in serum, milk, and tears after feeding Ascophyllum nodosum to dairy cows-A pilot study.

Kelp (Ascophyllum nodosum) is rich in iodine and often fed by organic dairy producers as a mineral supplement to support animal health. A commonly held belief is that kelp supplementation decreases susceptibility to infectious bovine keratoconjunctivitis due to increased iodine concentrations in tears. Whereas serum and milk iodine concentrations are positively correlated and modulated by oral iodine supplementation, nothing is known about the iodine concentration of tears. Therefore, the 3 objectives of this pilot study were to determine (1) the iodine content of tears, milk, and serum of cows after being fed kelp for 30d; (2) the trace mineral and thyroid status of cows before (d 0) and after being fed kelp for 30d; and (3) the in vitro growth rate of bacteria in tears (Moraxella bovis) or milk (Staphylococcus aureus, Escherichia coli, Streptococcus uberis) collected from cows fed no kelp (d 0) or kelp (d 30). Cows (n=3/treatment) were individually fed 56g of kelp per day (n=3/treatment) or not (n=3/no treatment) for 30 d. Daily feed intake of the TMR was recorded and weekly TMR, kelp, milk, blood and tear samples were collected and analyzed for iodine. The feed samples were pooled and further analyzed for other minerals. On d 0 and 30, liver biopsies and blood samples were collected and analyzed for mineral content and thyroid hormone concentrations, respectively. An inhibition test used milk and tear-soaked plates from kelp-fed cows (d 0 and 30) as well as 1 and 7.5% iodine as positive and distilled water as negative control. As expected, serum iodine concentrations were positively correlated with milk and tear iodine concentrations. Whereas the iodine concentrations in serum increased significantly in the kelp-fed cows during the 30-d study, milk and tear iodine concentrations increased only numerically in these cows compared with the control group. Liver mineral profiles were comparable between groups and generally did not change over the course of the study. Thyroid hormones remained overall within the reference range throughout the trial. Neither milk nor tears from kelp-fed cows inhibited in vitro growth of any of the plated bacteria. In summary, serum iodine concentration was correlated with the iodine concentration in milk and tears and feeding kelp increased only the serum iodine levels of cows in this trial. Bacterial growth was not inhibited in milk and tears of kelp-fed cattle in vitro, and prevention of infectious bovine keratoconjunctivitis would not be based solely on increased iodine concentrations in tears.

Conjunctival flora, Schirmer's tear test, intraocular pressure, and conjunctival cytology in neotropical primates from Salvador, Brazil.

BACKGROUND: This study aimed to establish reference values for selected ophthalmic diagnostic tests in healthy neotropical primates from Salvador, Brazil. METHODS: A total of 73 intact adults, including Callithrix jacchus (n = 31), Callithrix penicillata (n = 8), Cebus sp. (n = 22), and Cebus xanthosternos (n = 9) were used to evaluate the normal conjunctival bacterial flora. Cebus xanthosternos (n = 12) were used to evaluate tear production with Schirmer's tear test (STT), intraocular pressure (IOP), and conjunctival cytology. RESULTS: For all animals evaluated, Gram-positive bacteria were predominant. Results of the diagnostic tests in Cebus xanthosternos were as follows: STT: 14.92 ± 5.46 mm/minutes, IOP: 19.62 ± 4.57 mmHg, and conjunctival cytology revealed intermediate squamous epithelial cells in great quantities. CONCLUSIONS: These ophthalmic reference values will be particularly useful to diagnose discrete or unusual pathological changes in the neotropical primates eye.

Ocular Surface Microbiota in Diabetic Patients With Dry Eye Disease.

Purpose: To investigate the ocular surface (OS) commensal bacteria profiles of patients with diabetes mellitus (DM) and dry eye disease (DED). Methods: In the present study, subjects were assigned to four groups: 37 to the diabetic mellitus with dry eye disease (DM with DED) group, 22 to the diabetes mellitus (DM)-only group, 34 to the dry eye disease (DED)-only group, and 22 to the control group. Tear fluid was collected using Schirmer's tear secretion test paper. 16S ribosomal ribonucleic acid (rRNA) gene sequencing was used to analyze the bacterial microbiota. Results: The DM with DED group showed the highest operational taxonomic unit (OTU) numbers and alpha diversity and the most different beta diversity. The groups shared the four most abundant phyla, accounting for over 96% of the total abundance. At the genus level, there were 10 types of overlap in the core microbiota in the groups. They showed significant differences between the groups. Additionally, the DM with DED group and the control group showed four unique core genera, respectively. Unclassified Clostridiales and Lactobacillus were the core microbiota members of the DM with DED group, the DM-only group, and the DED-only group, but not the control group. Conclusions: In the present study, our results showed that the patients in the DM with DED group had a more complex and comprehensive ocular surface microbial composition. To the best of our knowledge, this is the first study to reveal the microbial profile of dry eye disease in patients with diabetes mellitus.

The closed eye harbours a unique microbiome in dry eye disease.

Dry eye affects millions of individuals. In experimental models, dry eye disease is associated with T helper cell 17-mediated inflammation of the ocular surface that may cause persistent damage to the corneal epithelium. However, the initiating and perpetuating factors associated with chronic inflammation of the ocular surface remain unclear. The ocular microbiota alters ocular surface inflammation and may influence dry eye disease development and progression. Here, we collected serial samples of tears on awakening from sleep, closed eye tears, during a randomized clinical trial of a non-pharmaceutical dry eye therapy and used 16S rRNA metabarcoding to characterize the microbiome. We show the closed dry eye microbiome is distinct from the healthy closed eye microbiome, and that the microbiome remains distinct despite daily saline eye wash upon awakening. The ocular microbiome was described only recently, and this report implicates a distinct microbiome in ocular disease development. Our findings suggest an interplay between microbial commensals and inflammation on the ocular surface. This information may inform future studies of the pathophysiological mechanisms of dry eye disease.

Metagenomic Profiling of Ocular Surface Microbiome Changes in Meibomian Gland Dysfunction.

Purpose: Ocular surface microbiome changes can affect meibomian gland dysfunction (MGD) development. This study aimed to delineate differences among the microbiome of eyelid skin, conjunctiva, and meibum in healthy controls (HCs) and patients afflicted with MGD. Methods: Shotgun metagenomic analysis was used to determine if there are differences between the microbial communities in ocular sites surrounding the meibomian gland in healthy individuals and patients afflicted with MGD. Results: The meibum bacterial content of these microbiomes was dissimilar in these two different types of individuals. Almost all of the most significant taxonomic changes in the meibum microbiome of individuals with MGD were also present in their eyelid skin, but not in the conjunctiva. Such site-specific microbe pattern changes accompany increases in the gene expression levels controlling carbohydrate and lipid metabolism. Most of the microbiomes in patients with MGD possess a microbe population capable of metabolizing benzoate. Pathogens known to underlie ocular infection were evident in these individuals. MGD meibum contained an abundance of Campylobacter coli, Campylobacter jejuni, and Enterococcus faecium pathogens, which were almost absent from HCs. Functional annotation indicated that in the microbiomes of MGD meibum their capability to undergo chemotaxis, display immune evasive virulence, and mediate type IV secretion was different than that in the microbiomes of meibum isolated from HCs. Conclusions: MGD meibum contains distinct microbiota whose immune evasive virulence is much stronger than that in the HCs. Profiling differences between the meibum microbiome makeup in HCs and patients with MGD characterizes changes of microbial communities associated with the disease status.

Clearance of Pseudomonas aeruginosa from a healthy ocular surface involves surfactant protein D and is compromised by bacterial elastase in a murine null-infection model.

Our previous studies showed that surfactant protein D (SP-D) is present in human tear fluid and that it can protect corneal epithelial cells against bacterial invasion. Here we developed a novel null-infection model to test the hypothesis that SP-D contributes to the clearance of viable Pseudomonas aeruginosa from the healthy ocular surface in vivo. Healthy corneas of Black Swiss mice were inoculated with 10(7) or 10(9) CFU of invasive (PAO1) or cytotoxic (6206) P. aeruginosa. Viable counts were performed on tear fluid collected at time points ranging from 3 to 14 h postinoculation. Healthy ocular surfaces cleared both P. aeruginosa strains efficiently, even when 10(9) CFU was used: e.g., <0.01% of the original inoculum was recoverable after 3 h. Preexposure of eyes to bacteria did not enhance clearance. Clearance of strain 6206 (low protease producer), but not strain PAO1 (high protease producer), was delayed in SP-D gene-targeted (SP-D(-/-)) knockout mice. A protease mutant of PAO1 (PAO1 lasA lasB aprA) was cleared more efficiently than wild-type PAO1, but this difference was negligible in SP-D(-/-) mice, which were less able to clear the protease mutant. Experiments to study mechanisms for these differences revealed that purified elastase could degrade tear fluid SP-D in vivo. Together, these data show that SP-D can contribute to the clearance of P. aeruginosa from the healthy ocular surface and that proteases can compromise that clearance. The data also suggest that SP-D degradation in vivo is a mechanism by which P. aeruginosa proteases could contribute to virulence.

Recurrent herpes in the rabbit and man.

Herpesvirus was present in secretory glands and frequently in tears of rabbits with recurrent herpetic keratitis even in the absence of corneal lesions. In normal people, herpesvirus could be cultured from saliva and tears. Chronic virus multiplication in structures such as the lacrimal and salivary glands, rather than latency, may cause recurrent herpetic disease.

Label-free electrical sensing of bacteria in eye wash samples: A step towards point-of-care detection of pathogens in patients with infectious keratitis.

The diagnosis of keratitis is based on visual exam, tissue cytology, and standard microbial culturing to determine the type of the infectious pathogen. To prescribe appropriate therapy, it is important to distinguish between bacterial, fungal, and viral keratitis, as the treatments are quite different. Diagnosis of the causative organism has a substantial prognostic importance. Further, timely knowledge of the nature of the pathogen is also critical to adapt therapy in patients unresponsive to empiric treatment options, which occurs in 10% of all cases. Currently, the identification of the nature of the pathogen that causes keratitis is achieved via microbial culture screening, which is laboratory-based, expensive, and time-consuming. The most frequent pathogens that cause the corneal ulcers are P. aeruginosa and S. aureus. Here, we report a microchip for rapid (<1h) detection of P. aeruginosa (6294), S. aureus(LAC), through on-chip electrical sensing of bacterial lysate. We evaluated the microchip with spiked samples of PBS with bacteria concentration between 101 to 108 CFU/mL. The least diluted bacteria concentration in bacteria-spiked samples with statistically significant impedance change was 10 CFU/mL. We further validated our assay by comparing our microchip results with the standard culture-based methods using eye washes obtained from 13 infected mice.

Methods for In Vitro Analysis of Antimicrobial Activity and Toxicity of Anti-keratitis Peptides: Bacterial Viability in Tears, MTT, and TNF-α Release Assays.

Ease of access to the cornea makes antimicrobial peptides (AMPs) ideal candidates for topical drug application. However, before bringing them to the clinic, it is fundamental to evaluate in vitro: (1) the ability of AMPs to kill bacteria in the presence of human tears, by counting the number of surviving bacteria on agar plates; (2) the potential cytotoxicity of AMPs to mammalian cells by a colorimetric method based on the production of a colored formazan crystals by metabolically active cells; and (3) the ability of AMPs to neutralize the toxic effect of the bacterial cell wall component, lipopolysaccharide (LPS), by measuring the level of the pro-inflammatory cytokine, TNF-α, released from LPS-activated macrophages, using a sandwich enzyme-linked immunosorbent assay.

Helicobacter pylori in lacrimal secretions.

The aim of this study was to investigate the presence of Helicobacter pylori in human lacrimal and nasal secretions. Eighty patients with complaints of dyspepsia who had undergone endoscopies and gastric antrum biopsies were included in the study. A total of five specimens, including 2 lacrimal secretion samples, 2 nasal mucosal swab samples, and 1 gastric antrum biopsy, were collected from each patient and investigated with polymerase chain reaction (PCR) methods consisting of the urease enzyme coding gene GlmM (UreC) and the H pylori-specific 16S rRNA coding gene. The Reflux Symptom Index and ophthalmologic complaints of the patients were recorded. The detected positivity rates of the H pylori 16S rRNA coding gene in gastric biopsies and nasal mucous and lacrimal secretions were 55, 11.2, and 20%, respectively. The patients were grouped as gastric-antrum-biopsy-negative (Group I [n = 36]) and -positive (Group II [n = 44). In Group II, H pylori positivity in the lacrimal and nasal mucous secretions was 36.3 and 18%, respectively. A comparison between the groups in terms of H pylori presence in nasal mucous and lacrimal secretions yielded statistically significant differences (p = 0.0001, p = 0.003). The simultaneous presence of H pylori in nasal mucous and lacrimal secretions was 13.6% in Group II. H pylori positivity in nasal mucous and lacrimal secretions had a positive moderate correlation (r = 0.40; p = 0.0003). The present study is the first report on the presence of H pylori in lacrimal secretions through nested PCR, which suggested the presence of a number of mechanisms for H pylori transmission to lacrimal secretions.

Neurovirulence of two clonally related herpes simplex virus type 1 strains in a rabbit seizure model.

Herpes simplex virus type 1 (HSV-1) strains vary widely with regard to neurovirulence, but their tropism for specific central nervous system structures and their ability to induce seizures are poorly defined. We have used the clonally related +GC and -GC strains of HSV-1 to define the pathophysiological basis of neurovirulence in a rabbit model. Following intranasal inoculation, +GC infection was nearly uniformly fatal while -GC infection was asymptomatic. The +GC infected animals developed electroencephalographic (EEG) abnormalities which preceded severe motor seizures. Tropism of the +GC strain for specific CNS nerve centers and the expression of viral antigens within them correlated with its virulence. Although both viruses invaded and replicated within the brain, +GC replicated to slightly higher titers and expressed more abundant viral antigen than -GC. The relatively less efficient replication of -GC appeared to correlate with its temperature-sensitive phenotype in vitro. Both +GC and -GC antigens were found in cerebral cortical layers IV-VI, and in several central nervous system trigeminal and olfactory system structures. However, +GC spread more completely throughout the brain to involve the amygdala, nucleus accumbens, several brainstem nuclei and the locus ceruleus. The +GC antigens were also found in cerebral cortical layer I of animals that developed seizures. These results indicate that the ability of HSV-1 to induce electrophysiologic brain abnormalities is associated with its ability to replicate within specific brain nerve centers.

Quantitation of herpes simplex virus in vitro and in preocular tear film.

Current methods for virus isolation from preocular tear film are not quantitative. This report presents a sensitive method for detection and quantitation of herpes simplex virus type 1 in unknown samples of small size (less than 50 microliters). Serial tenfold dilutions of stock virus were inoculated onto 16 mm vero cell monolayers, which were monitored for the development of cytopathologic evidence (CPE) for presence of virus and were assigned a severity grade. Standard curves were developed on the basis of time interval between inoculation, recognition of CPE, and CPE score. These curves were used to predict virus titers in unknown samples. This method for virus isolation is simple, efficient, and consistently sensitive to virus titers of less than 10 plaque forming units.

Conjunctivitis in rabbits caused by enterovirus type 70 (EV70).

A rabbit enterovirus 70 (EV70) model infection that closely mimics human enteroviral conjunctivitis was developed. Conjunctivitis occurred 24 hr following topical application of EV70. The conjunctivitis was characterized by tearing, redness, swelling of the eye lids, follicles in the superior palpebral conjunctiva, and dilatation of subconjunctival blood vessels. Histologic examination of conjunctival and corneal tissue taken 1 and 2 days after infection revealed numerous punctate areas devoid of squamous epithelium on the upper palpebral conjunctiva. Also, follicles without germinal centers were observed microscopically in the palpebral and tarsal conjunctiva. Fibroblast infiltration characteristic of wound healing and a sparse mononuclear infiltration was noted by the second day. Peak levels of virus [10(3) to 10(6.2) plaque forming units (PFU)/ml] were detected 1 to 2 days after infection and declined to undetectable levels after 3 to 5 days. Interestingly, antiserum to parental EV70 was less effective (8-10-fold) in neutralizing EV70 adapted to animal and tissue culture systems. This finding suggests that an antigenic variant of EV70 arose during adaptation. Fibroblast interferon (IFN beta), which is indicative of viral infection, was detected in tears from 6 of 16 rabbits and declined to undetectable levels 3 days after infection. Serum antibody to EV70 was detectable 8 to 10 days after infection. However, the level of serum antibody was highly variable. The results indicate that the clinical disease, virologic and immunologic courses were similar to that of the human infection. Results suggest that this animal model provides a system for studying the natural antigenic variation of EV70, the natural host defenses of the eye, and antiviral treatments against enteroviral conjunctivitis.

HSV-1 recovery from ocular tissues after viral inoculation into the superior cervical ganglion.

New Zealand albino rabbits were inoculated in the right superior cervical ganglion with 25 microliter of herpes simplex virus type 1 (HSV-1) (McKrae strain; 10(3) or 10(5) PFU/ml). Positive tear film swabs were detected at least once in 28/32 (88%) of ipsilateral eyes and 6/32 (19%) of contralateral eyes beginning on postinoculation (PI) day 2-6. The average HSV-1 titer in the tear film was 4.0 X 10(3) PFU in ipsilateral eyes and 2.7 X 10(3) PFU in contralateral eyes, determined from eye washes after inoculation of 25 PFU of HSV-1. In selected rabbits, the aqueous humor was positive for virus on PI days 3, 4, 5, 6, and 8. the aqueous humor in ipsilateral eyes showed positive results in 9/11 (82%) of the eyes tapped on PI 3, 13/18 (72%) on PI 4, 5/11 (45%) on PI 5, 1/6 (17%) on PI 6, and 1/2 (50%) on PI 8. No virus was detected in aqueous humor tappings in any contralateral eyes (0/65). Conjunctivitis and iritis (iris hyperemia) appeared in all ipsilateral eyes beginning as early as PI day 1. Conjunctivitis occurred in 1/21 (4.8%) of contralateral eyes. Cells and flare appeared in 18/21 (86%) of ipsilateral eyes and 2/21 (9.5%) of contralateral eyes. Hyphema was noted in 3/21 (14%) of ipsilateral eyes. Of the eyes with iritis, 12/21 (57%) developed corneal edema. Corneal dendritic ulcers were observed in 4/21 (19%) of ipsilateral eyes and 2/21 (9.5%) of contralateral eyes. No ocular fundus changes were seen in any contralateral or ipsilateral eyes.(ABSTRACT TRUNCATED AT 250 WORDS)

Herpes simplex virus recovery in neural tissues after ocular HSV shedding induced by epinephrine iontophoresis to the rabbit cornea.

Ocular HSV-1 shedding from latently infected rabbits was induced by iontophoresis of 0.01% epinephrine into the eye. Anodal Iontophoresis of epinephrine was performed at 0.8 mAmps for 8 min once a day for 3 consecutive days. Shedding was determined by the presence of HSV-1 in the preocular tear film obtained via eye swabs. Bilateral epinephrine iontophoresis performed on selected days during 220-280 days after inoculation resulted in HSV-1 shedding in 75% of the eyes (30/40) and 100% of the rabbits (20/20). Following the induction of ocular HSV-1 shedding, rabbits were killed and selected neural tissues were homogenized. Cell-free preparations were assayed for the presence of infectious virions using primary rabbit kidney cell monolayers. When the tissues were homogenized immediately after death, virus was detected in only one neural tissue, the trigeminal ganglia. However, when the tissues were incubated in vitro for 18-24 hours prior to the homogenization, infectious HSV-1 was recovered from homogenates of the trigeminal ganglion, superior cervical ganglion, the ophthalmic branch of the trigeminal nerve, and the root entry zone of the trigeminal nerve. A relationship was noted between the time of the last ocular shedding and recovery of infectious HSV from the tissue homogenates. Furthermore, a positive correlation in 11 eyes between the recovery of HSV-1 from the perocular tear film and HSV-1 recovery from one or more corresponding neural tissues was found. These results suggested that epinephrine iontophoresis to the cornea triggered an "alteration" in the state of the virus in the neural tissues of the latently infected rabbits and that the change can be related to the induced ocular shedding.