Diversity of virulence-associated genes in pathogenic Aeromonas hydrophila isolates and their in vivo modulation at varied water temperatures.

Most environmental parameters have no consistent effect on the expression of bacterial genes responsible for their virulence. However, as fish are poikilothermic, the possibility of temperature variation having a pronounced effect on the expression of virulence-associated gene(s) of bacteria infecting the host needs to be investigated. In this study, the diversity of virulence genes in seven Aeromonas hydrophila isolates collected from diseased fish from different parts of India was characterized, and the effect of temperature variation on the extent of expression of their virulence was investigated. All bacterial isolates were screened for a total of nine bacterial virulent genes {aerolysin, hemolysin, cytoen, outer membrane protein TS (Omp TS), elastase, flagellin, lipase, ß hemolysin and type 3 secretion system}, and the diversity in their presence or absence were marked at a particular in vitro condition. Three bacterial isolates (nos. 1, 7 and 2) were selected for further study, based on their ability to cause varied mortalities (20-100%) in Labeo rohita juveniles in intraperitoneal challenge study. Further, three isolates were injected intraperitoneally into L. rohita fingerlings at three different temperatures (i.e., 20, 28 and 37 °C) and at 6 h post-challenge, the kidney samples were collected to measure the levels of all nine bacterial virulence genes using semi-quantitative PCR. The maximum level of amplicons of virulence genes in all three A. hydrophila isolates was noticed at 28 °C as compared to 37 °C and 20 °C. It was also observed that haemolysin played a more prominent role in the expression of virulence, when compared to cytoen gene. Hence, it was concluded that water temperature does play a crucial role in governing virulence gene expression, and a temperature of 28 °C would be considered as suitable for looking into the pathogenicity of A. hydrophila for conducting any challenge study with this organism in tropical environment.

Dynamics of expression of antibacterial and antioxidant defence genes in Indian major carp, Labeo rohita in response to Aeromonas hydrophila infection.

Cells produce large number of antioxidant molecules to prevent reactive oxygen species-induced self-damage during microbial assault while generating simultaneously number of antimicrobial molecules to target the pathogen. The present study was aimed at looking into molecules involved in antibacterial and self-protection mechanism of a host Labeo rohita when challenged with a pathogenic bacterium Aeromonas hydrophila. Expression profiles of few of the important host antibacterial genes viz., inducible nitric oxide synthase (iNOS), lysozyme G (LysoG), apolipoprotein A-I (ApoA-I) and hepcidin, and self-defence anti-oxidant genes viz., manganese superoxide dismutase (MnSOD), catalase and glutathione peroxidases (GPx3) were examined in skin and muscle tissues of bacteria challenged fish. Transcription levels of iNOS, LysoG, ApoA-I, hepcidin, catalase, GPx3 and MnSOD were significantly upregulated (P < 0.05) in both tissues at different time points post-bacterial challenge. Increased expression of antibacterial genes in the muscle and skin clearly explains strong defensive mechanism activated in fish tissues in terms of both oxygen-dependent (iNOS) and independent (lysozyme) ways of microbe reduction, and bacterial lysis via production of antimicrobial molecules (ApoA-I and hepcidin) in the host. Simultaneous upregulation of MnSOD, GPx3 and catalase genes explains their involvement in patrolling the cells with regulated production of reactive oxygen species and keeping at a safe level to protect the host's own cells from oxidative damage.

Effect of depth of the sclerocorneal incision on postoperative corneal astigmatism in manual small-incision cataract surgery.

Purpose: To determine the effect of depth of scleral tunnel incision measured by anterior segment OCT on postoperative corneal astigmatism by comparing the change of magnitude of corneal astigmatism between superficial and deep sclerocorneal tunnel incision in manual small-incision cataract surgery (SICS). Methods: Depths of sclerocorneal incision of 72 eyes of patients undergoing uncomplicated manual SICS and attending regular follow-up schedule were assessed with anterior segment OCT at 6-week post-op follow-up. Results: The overall mean ± standard deviation (SD) change of astigmatism for superficial incision, that is, ≤399 µm, was 0.44 ± 0.30 and that for deeper, that is, ≥400 µm, was 0.13 ± 0.48 and the change was significantly higher in ≤399 µm group than in ≥400 µm group (P = 0.003). In both superior and temporal incision locations, the mean ± SD change of astigmatism for ≤399 µm incision was 0.48 ± 0.29 and 0.40 ± 0.30, respectively, and that for ≥400 µm was 0.03 ± 0.34 and 0.23 ± 0.57, respectively. The change of astigmatism was significantly higher in ≤399 µm incision group overall (P = 0.003) and also higher in both superior and temporal incision location groups (P = 0.001 and P = 0.479, respectively). Conclusion: The depth of sclerocorneal incision had a statistically significant effect on the change of astigmatism following manual SICS, with superficial incision (≤399 µm) causing a higher change than deeper incision (≥ 400 µm).

Effectiveness of a modified therapeutic protocol for the management of vernal keratoconjunctivitis based on Bonini's graded clinical severity.

Purpose: To evaluate the effectiveness of a modified therapeutic protocol used for vernal keratoconjunctivitis (VKC) based on severity as per Bonini grading system. Methods: This was a prospective observational clinical study conducted with 123 eyes of 63 patients. A meticulous clinical examination was performed, and data was documented in all the cases. Patients on known systemic atopy and antiallergic therapy were excluded from the study. Eyes with a clinical diagnosis of VKC were segregated based on Bonnini's grading system. A treatment protocol was created depending on the grade of VKC. Therapeutic responses were documented at 3 weeks, 3 months, 6 months, 12 months, and 24 months. Grading of the eyes was performed in each visit. Results: The mean age of the patients was 8.85 years with a standard deviation of 4.48 years. Males were predominant, and 95.24% had bilateral manifestation. The palpebral component was the most common form of manifestation. Itching was the most common manifestation, followed by congestion, discharge, and papillae in a decreasing order. Also, 68% of patients were in grade 2, 14% in grade 3, 12% in grade 1, and the rest were in grade 4. Following the treatment protocol, 70% showed signs of significant improvement in grade by the end of 6 weeks, reaching 90% at the end of 6 months (P = 0.074) and 92% at the end of 12 months (P = 0.002). Also, 52.4% versus 77.8% of patients had no recurrence in the pre- versus posttreatment protocol and it was statistically significant (P = 0.001). Conclusion: Grading of VKC gives a clear evaluation of the severity and progression of the condition. Besides, significant improvement in the grades was observed with fewer incidences of recurrences following execution of the therapeutic protocol. Hence, it is essential to maintain a treatment protocol in our clinical practice to provide grade-based therapy and monitor accurate changes in the clinical condition.

Transconjunctival botulinum toxin injection into the lacrimal gland in crocodile tears syndrome.

Purpose: To determine the efficacy and safety of botulinum toxin injection into the lacrimal gland as a symptomatic treatment of crocodile tear syndrome (CTS). Methods: Our study included six patients of unilateral gustatory hyper lacrimation following either an episode of facial paralysis or post trauma or any related surgery that posed a risk of damaging the facial nerve. Detailed history regarding previous trauma, duration of facial paralysis, previous significant surgery, and duration of steroid use following facial paralysis was noted. Schirmer's test was done at baseline and 3 months follow-up. Patients' consent was taken prior to treatment with botulinum toxin injection in the lacrimal gland. Repeat injection of 5 U botulinum toxin (type A) was administered into the lacrimal gland of all patients transconjunctivally within an interval of 1 week. All patients were followed up with Schirmer's test at 6 weeks and 3 months. Any complications during treatment were recorded. Results: All six patients showed complete or partial disappearance of reflex lacrimation while chewing following botulinum injection measured by a significant reduction in Schirmer's value. When comparing Schirmer test values before (27.8 ± 3.58 mm) and after (11.6 ± 2.28 mm) BTX-A injection, the differences observed (P = 0.002) were statistically significant (P < 0.05). Only two patients developed mild transitory ptosis. No other complications were noted. Conclusion: Transconjunctival botulinum toxin injection into the lacrimal gland is an effective and safe method to decrease reflex lacrimation during eating or chewing in CTS or gustatory hyper-lacrimation syndrome.

Postoperative astigmatic considerations in manual small-incision cataract surgery - A review.

Cataract remains a major cause of visual impairment worldwide including in India. The sutureless manual small-incision cataract surgery (MSICS) as an alternative to phacoemulsification, gives equivalent visual results at lower expenses. Still the procedure is often discredited for higher astigmatism due to the larger size of the incision. High astigmatism is an important cause of poor uncorrected visual acuity after cataract surgery. However, there are enough studies in the literature to prove that surgically induced astigmatism (SIA) can be minimized and also eliminated by adopting appropriate wound construction techniques during surgery. Even pre-existing astigmatism if any can be neutralized by changing wound architecture during surgery. Here, we review the various techniques of scleral tunnel construction described in the literature to care for postoperative astigmatism in MSICS.

Stabilization of refraction and timing of spectacle prescription following manual small-incision cataract surgery.

Purpose: To determine the stabilization of refraction at 2 weeks following MSICS by comparing the difference in spherical, cylindrical component and also spherical equivalent of refraction of 2 weeks follow-up with that of 6 weeks following surgery. Methods: The difference of spherical, cylindrical component and also spherical equivalent of refraction at 2 weeks and 6 weeks follow-up of 194 eyes that underwent uncomplicated MSICS with implantation of PMMA IOL conducted by a single experienced surgeon were compared to find out the amount of change and its significance was statistically tested by Wilcoxon-Signed Rank Test. Results: The difference in spherical power (0.04 ± 0.30), cylinder power (0.03 ± 0.40), and spherical equivalent (0.06 ± 0.34) were very small and not significant statistically (P-value ≤0.05). Conclusion: Necessary spectacle correction can safely be prescribed after 2 weeks following MSICS as subjective refraction stabilizes by that time without undergoing significant change. However, our observation was applicable in patients who had an uneventful cataract surgery without any risk factor, which can delay wound healing or cause poor visual outcome.

Toxic non-inflammatory fungal keratitis.

Purpose: To report a new entity called "toxic non-inflammatory fungal keratitis." Methods: Eyes manifesting infective keratitis with a history of prior administration of topical steroids were included in the study. The details pertaining to the type of injury, duration of injury, and primary treatment for corneal trauma were meticulously documented. The corneal tissues were scraped from the patients and were analyzed for fungal filaments by using a 10% KOH mount under a compound microscope. Moreover, these scraped materials were plated on blood agar and Sabouraud dextrose agar plates. Results: The corneal ulcers displayed a disproportionately reduced intensity of pain and improved visual acuity. Further, 10% KOH revealed profuse fungal filaments with few inflammatory cells in all the patients. The anterior chamber cells and flare were either reduced or entirely absent. There was no evidence of lid edema and surrounding corneal edema in any of the patients. The mean healing period was 28.8 days (standard deviation (SD): 10.05). The KOH mount revealed the presence of confluent fungal hyphae with a few inflammatory cell infiltrates. The Aspergillus species and Fusarium species were found in 47% and 40% of the cases, respectively. Conclusion: Toxic non-inflammatory fungal keratitis following steroid therapy needs to be considered in fungal ulcers with disproportionately less pain and good visual acuity. The fungal ulcers with altered clinical signs of classical inflammation need to be assessed for topical steroid misuse.

Comodulation of Dengue and Chikungunya Virus Infection During a Coinfection Scenario in Human Cell Lines.

The Dengue virus (DENV) and Chikungunya virus (CHIKV) are the arboviruses that pose a threat to global public health. Coinfection and antibody-dependent enhancement are major areas of concern during DENV and CHIKV infections, which can alter the clinical severity. Acute hepatic illness is a common manifestation and major sign of disease severity upon infection with either dengue or chikungunya. Hence, in this study, we characterized the coexistence and interaction between both the viruses in human hepatic (Huh7) cells during the coinfection/superinfection scenario. We observed that prior presence of or subsequent superinfection with DENV enhanced CHIKV replication. However, prior CHIKV infection negatively affected DENV. In comparison to monoinfection, coinfection with both DENV and CHIKV resulted in lower infectivity as compared to monoinfections with modest suppression of CHIKV but dramatic suppression of DENV replication. Subsequent investigations revealed that subneutralizing levels of DENV or CHIKV anti-sera can respectively promote the ADE of CHIKV or DENV infection in FcγRII bearing human myelogenous leukemia cell line K562. Our observations suggest that CHIKV has a fitness advantage over DENV in hepatic cells and prior DENV infection may enhance CHIKV disease severity if the patient subsequently contracts CHIKV. This study highlights the natural possibility of dengue-chikungunya coinfection and their subsequent modulation in human hepatic cells. These observations have important implications in regions where both viruses are prevalent and calls for proper management of DENV-CHIKV coinfected patients.

Japanese Encephalitis Virus NS4A Protein Interacts with PTEN-Induced Kinase 1 (PINK1) and Promotes Mitophagy in Infected Cells.

The nonstructural protein 4A (NS4A) of flaviviruses has been implicated as a "central organizer" of the membrane-bound replication complex during virus replication. However, its role in the host responses to virus infection is not understood. Using the yeast-two-hybrid library screen, we identified a multitude of host proteins interacting with the Japanese encephalitis virus (JEV) NS4A protein. Several of these interacting proteins are known to localize to the mitochondria. One of these proteins was PTEN-induced kinase 1 (PINK1), a serine/threonine-protein kinase known for its role in mitophagy. Here, we demonstrate the JEV-NS4A localization to the mitochondria and its interaction with PINK1 in Huh7 cells during JEV infection. The JEV-infected cells showed an enhanced mitophagy flux with a concomitant decline in the mitochondrial mass. We present data showing that JEV-NS4A alone was sufficient to induce mitophagy. Interference with mitochondrial fragmentation and mitophagy resulted in reduced virus propagation. Overall, our study provides the first evidence of mitochondrial quality control dysregulation during JEV infection, largely mediated by its NS4A protein. IMPORTANCE The JEV-infected mammalian cells show an enhanced mitophagy flux with a concomitant decline in the mitochondrial mass. We show that the NS4A protein of JEV localized to the mitochondria and interacted with PINK1 in Huh7 cells during infection with the virus and demonstrate that JEV-NS4A alone is sufficient to induce mitophagy. The study provides the first evidence of mitochondrial quality control dysregulation during JEV infection, largely mediated by its NS4A protein.

Universal eye health in Odisha, India, Sunetra. Report # 1. Program planning.

PURPOSE: To describe the program planning of Sunetra (Healthy Eyes), the universal eye health coverage in the Indian state Odisha. METHODS: The Indian state of Odisha launched the universal eye health coverage in October 2017. This is the first Indian state to officially respond to the World Health Organization's Global Action Plan. RESULTS: The Government of Odisha pledged INR 6,820 million (US$ 100 million approximately) spread over 5 years. Each third of the pledged money is allocated for immediate direct benefit, building the infrastructure and human resources, and creating a digital eye health platform for uniform documentation and policy planning. CONCLUSION: It is hoped that at the end of 5 years, by 2022, the state will attain the goals prescribed in the Global Action Plan in eye care.