Unveiling the ocular battlefield: Insights into Pseudomonas aeruginosa virulence factors and their implications for multidrug resistance.

The research investigates the virulence factors of Pseudomonas aeruginosa (P. aeruginosa), a pathogen known for its ability to cause human infections by releasing various exoenzymes and virulence factors. Particularly relevant in ocular infections, where tissue degeneration can occur, even after bacterial growth has ceased due to the potential role of secreted proteins/enzymes. Clinical isolates of P. aeruginosa, both ocular (146) and non-ocular (54), were examined to determine the frequency and mechanism of virulence factors. Phenotypic characterization revealed the production of alginate, biofilm, phospholipase C, and alkaline protease, while genotypic testing using internal uniplex PCR identified the presence of Exo U, S, T, Y, and LasB genes. Results showed a significant prevalence of Exo U and Y genes in ocular isolates, a finding unique to Indian studies. Additionally, the study noted that ocular isolates often contained all four secretomes, suggesting a potential link between these factors and ocular infections. These findings contribute to understanding the pathogenesis of P. aeruginosa infections, particularly in ocular contexts, and highlights the importance of comprehensive virulence factor analysis in clinical settings.

Role of polymerase chain reaction-based viral detection in pterygia.

Purpose: Pterygium is a fibrovascular disease that originates in the conjunctiva and commonly spreads to the corneal surface, thereby posing a threat to eyesight. Despite intensive research, the pathophysiology of this disease remains unclear. Recent research suggests that oncogenic viruses, such as human papillomavirus (HPV), cytomegalovirus, and Epstein-Barr virus (EBV), may play a role in pterygia development. Although there are questions concerning the function of oncogenic viruses in pterygium pathogenesis, existing research shows a lack of consensus on the subject, demonstrating the heterogeneity of pterygium pathophysiology. Therefore, we aimed to simultaneously detect the three common viral pathogens that have been reported in pterygium tissue obtained after excision. Methods: Thirty-five tissue specimens of pterygium from patients undergoing pterygium surgery (as cases) were analyzed for evidence of viral infection with multiplex polymerase chain reaction (PCR), and virus-specific real-time quantitative PCR was used for the samples that were detected positive by multiplex PCR. Results: Of the 35 patients, one sample was positive for EBV and two samples were positive for HPV. Further PCR-based DNA sequencing of the HPV PCR-positive product showed identity with HPV-16. Real-time quantitative PCR on samples that showed EBV or HPV positivity did not yield any detectable copy number. Conclusion: Our study results confirmed that PCR positivity could be due to transient flora, but it was not quantitatively significant to conclude as the causative factor of pterygium pathogenesis. However, additional studies with larger sample populations are warranted to fully determine the role of the virus in pterygium.

Therapeutic Effect of Corneal Crosslinking on Fungal Keratitis: Efficacy of Corneal Collagen Crosslinking as an Adjuvant Therapy for Fungal Keratitis in a Tertiary Eye Hospital in South India.

PURPOSE: To evaluate the efficacy of CXL in treating fungal keratitis as an adjuvant therapy. METHODS: Detailed clinical examination microbiological investigation was performed. Twenty fungal keratitis patients were recruited and randomized into two groups: group 1 (n= 11, standard antifungal), group 2 (n=9, corneal collagen crosslinking with standard antifungal). Corneal scraping and tear samples collected were subjected to real-time PCR targeting ITS, TLR analysis and cytokine analysis. RESULTS: The mean time for complete resolution of ulcer for group 2 was significantly shorter compared to group 1 and the final mean BCVA was better for group 2. Expression of IL-1ß, IL-8, IFN-γ significantly decreased immediately post CXL in group 2 patients. Significant downregulation of TLR 6, TLR-3, TLR-4 was observed 3-days post CXL compared to group 1 patients. CONCLUSION: Adjuvant effect of CXL was significant in treating fungal keratitis compared to standalone antifungal treatment.

Author Correction: Genomic analysis of a large set of currently-and historically-important human adenovirus pathogens.

The original publication of this article [1] was missing the below author that made contributions to the research and the published article.

First Report on Detection of Dengue Virus in the Donor Cornea.

BACKGROUND: Dengue fever is the most prevalent form of flavivirus infection in humans. We have investigated whether corneoscleral tissue of the donor affected by dengue virus (DENV) harbors the virus. PURPOSE: To identify the risk for viral transmission through corneal transplants in areas where DENV circulates. METHODS: Excised corneoscleral tissue from a cadaver with a history of viral hemorrhagic fever was analyzed using reverse transcriptase-polymerase chain reaction for the presence of DENV and chikungunya virus (CHIV). RESULTS: DENV was detected in RNA extracted from the donor corneoscleral rim. Further genotyping of the viral isolate from the virus-infected cell harvest revealed DENV type 3 as the causative agent. CHIV was not detected. CONCLUSIONS: The data presented in this study recommend the implementation of polymerase chain reaction for detection of DENV and CHIV to analyze excised corneoscleral tissue of a donor with viral hemorrhagic fever.

Culture-positive unilateral panophthalmitis in a serology-positive case of dengue hemorrhagic fever.

Dengue fever, a mosquito-borne disease commonly found in the tropics, is one of the most prevalent forms of Flavivirus infection in humans. Symptomatically, it is characterized by fever, arthralgia, headache, and rash. Ophthalmic manifestations can involve both the anterior and posterior segment. Panophthalmitis is rare in dengue hemorrhagic fever, and there is no report of culture-positive panophthalmitis in this setting. Here, we report a case of a serology-positive 33-year-old male patient of dengue hemorrhagic fever who developed sudden onset pain, redness, and proptosis in the right eye. The patient subsequently developed panophthalmitis in his right eye, and Bacillus cereus was isolated from eviscerated sample. This case provides unique insights into pathogenesis of panophthalmitis in dengue and highlights the management options.

Genomic analysis of a large set of currently-and historically-important human adenovirus pathogens.

Human adenoviruses (HAdVs) are uniquely important "model organisms" as they have been used to elucidate fundamental biological processes, are recognized as complex pathogens, and are used as remedies for human health. As pathogens, HAdVs may effect asymptomatic or mild and severe symptomatic disease upon their infection of respiratory, ocular, gastrointestinal, and genitourinary systems. High-resolution genomic data have enhanced the understanding of HAdV epidemiology, with recombination recognized as an important and major pathway in the molecular evolution and genesis of emergent HAdV pathogens. To support this view and to actualize an algorithm for identifying, characterizing, and typing novel HAdVs, we determined the DNA sequence of 95 isolates from archives containing historically important pathogens and collections housing currently circulating strains to be sequenced. Of the 85 samples that were completely sequenced, 18 novel recombinants within species HAdV-B and D were identified. Two HAdV-D genomes were found to contain novel penton base and fiber genes with significant divergence from known molecular types. In this data set, we found additional isolates of HAdV-D53 and HAdV-D58, two novel genotypes recognized recently using genomics. This supports the thesis that novel HAdV genotypes are not limited to "one-time" appearances of the prototype but are of importance in HAdV epidemiology. These data underscore the significance of lateral genomic transfer in HAdV evolution and reinforce the potential public health impact of novel genotypes of HAdVs emerging in the population.

Effect of Lignocaine Concentration on Human Fibroblasts Growth in Eyes Undergoing Trabeculectomy: An in vitro Study.

PURPOSE: To evaluate the effect of lignocaine on growth and apoptosis indication of primary human Tenon's capsule fibroblast (HTFs) in an in vitro model. PATIENTS AND METHODS: Tenon's capsule tissue obtained from patients undergoing trabeculectomy were grown in cell culture medium. The effect of different concentrations of lignocaine (0.5, 1.0, 1.5, and 2%) on the morphology and growth of the fibroblasts was studied using microscopy, cell viability, and proliferation assay, and apoptosis was detected using the FITC Annexin V Apoptosis Kit. RESULTS: Morphological changes similar to those of apoptotic cells, including cytoplasmic vacuolation, shrinkage, and rounding were visualized in the cells treated with concentrations greater than 1.0% (i.e., 1.5, 2.0%). Though proliferation inhibition was found with all four concentrations (0.5-2.0%), the viability of cells decreased from 1.0% lignocaine. CONCLUSION: 0.5% lignocaine prevents proliferation of fibroblasts without causing apoptosis in vitro.

Virulence genome analysis of Pseudomonas aeruginosa VRFPA10 recovered from patient with scleritis.

Infectious keratitis is a major cause of blindness, next to cataract and majority of cases are mainly caused by gram negative bacterium Pseudomonas aeruginosa (P. aeruginosa). In this study, we investigated a P. aeruginosa VRFPA10 genome which exhibited susceptibility to commonly used drugs in vitro but the patient had poor prognosis due to its hyper virulent nature. Genomic analysis of VRFPA10 deciphered multiple virulence factors and P.aeruginosa Genomic Islands (PAGIs) VRFPA10 genome which correlated with hyper virulence nature of the organism. The genome sequence has been deposited in DDBJ/EMBL/GenBank under the accession numbers LFMZ01000001-LFMZ01000044.

Whole-Genome Sequencing and Mutation Analysis of Two Extensively Drug-Resistant Sputum Isolates of Mycobacterium tuberculosis (VRFCWCF XDRTB 232 and VRFCWCF XDRTB 1028) from Chennai, India.

We announce the draft genome sequence of two extensively drug-resistant Mycobacterium tuberculosis strains, VRFCWCF XDRTB 232 and VRFCWCF XDRTB 1028, isolated from the sputum samples of a patient clinically suspected to have tuberculosis, and we also report novel mutations that confer drug resistance.

Whole-Genome Sequencing of Streptomycin-Resistant Mycobacterium tuberculosis Isolate VRFCWCF MRTB 180 Reveals Novel and Potential Mutations for Resistance.

We announce the draft genome sequence of a streptomycin monoresistant Mycobacterium tuberculosis strain (VRFCWCF MRTB 180) isolated from sputum of a clinically suspected tuberculosis patient.

Whole Genome Sequence of Polyresistant Mycobacterium tuberculosis CWCFVRF PRTB 19 Sputum Isolate from Chennai, India, Closely Clustering with East African Indian 5 Genogroup.

We announce the draft genome sequence of a polyresistant Mycobacterium tuberculosis strain (CWCFVRF PRTB 19) isolated from the sputum of a clinically suspected tuberculosis patient, and it closely clusters to the East African Indian 5 (EAI5) lineage.

Comparative Genomic Analysis of Multidrug-Resistant Pseudomonas aeruginosa Clinical Isolates VRFPA06 and VRFPA08 with VRFPA07.

Pseudomonas aeruginosa isolates harboring acquired drug-resistant genes lead to increased mortality. Here, we have sequenced and annotated the genomes of two multidrug-resistant (MDR) P. aeruginosa isolates and a susceptible P. aeruginosa clinical isolate evidencing divergent antibiotic susceptibilities. Genomic analysis showed insight on the different genomic strategies adapted by P. aeruginosa to combat antimicrobial effects.

Isolation of Mycobacterium massiliense from a corneal biopsy in India.

INTRODUCTION: Rapidly growing mycobacteria (RGM) are ubiquitous and are usually considered as saprophytes, and have been recovered from the environment, particularly in dust, watery soil and water distribution systems. However, Mycobacterium massiliense is a rare causative agent of ocular infection. CASE PRESENTATION: We report a case of M. massiliense in a 44-year-old female with signs and symptoms of a corneal ulcer. We carried out PCR-based DNA sequencing targeting the hsp 65 gene for the identification of M. massiliense. To confirm the identification, we also performed PCR-based RFLP targeting the hsp65 gene and PCR-based DNA sequencing targeting the internal transcribed spacer region, which showed 97 % nucleotide identity with M. massiliense. CONCLUSION: To the best of our knowledge, this is the first study in India to report the detection of M. massiliense from a corneal biopsy.

Draft Genome Sequence of Multidrug-Resistant Pseudomonas aeruginosa Strain VRFPA02, Isolated from a Septicemic Patient in India.

Multidrug-resistant Pseudomonas aeruginosa strains, which are notable nosocomial pathogens, have greatly increased the mortality rate of septicemic patients due to treatment failure. Here, we report the draft genome sequence of P. aeruginosa strain VRFPA02, a human bloodstream isolate that has phenotypically proven to be resistant to a broad spectrum of antibiotics.

Optimization and application of a reverse transcriptase polymerase chain reaction to determine the bacterial viability in infectious endophthalmitis.

PURPOSE: To develop RNA based assay - reverse transcriptase polymerase chain reaction (RT-PCR) to detect viable bacteria in intraocular specimens obtained from patients with infectious endophthalmitis. MATERIALS AND METHODS: Thirty-five intraocular specimens (19 vitreous fluid and 16 aqueous humor) collected from patients with typical infectious endophthalmitis were subjected to conventional and molecular microbiological investigations. Culture negative, eubacterial genome PCR positive intraocular specimens were subjected to denaturing high performance liquid chromatography (dHPLC) for separation of mixed genomes and subsequently identified by PCR based DNA sequencing. In parallel, RT-PCR was performed to detect the presence of viable bacteria in intraocular specimens. RESULTS: Among 35 intraocular specimens, single bacterial genome was detected in 9 (25.7%) and two or more genomes in 26 (74.28%) intraocular specimens. Eubacterial genome was detected by RT-PCR in 29 (82.85%) specimens. PCR based dHPLC followed by PCR based DNA sequencing revealed the presence of 65 bacterial genomes in 35 intraocular specimens. Five novel genera namely Terrabacter species, Facklamia species, Xylella fastidiosa, Duganella species and Synechococcus species were detected. CONCLUSION: RT-PCR serves as a rapid and reliable tool to detect viable bacteria causing endophthalmitis.

Phylogenetic comparison of exonic US4, US7 and UL44 regions of clinical herpes simplex virus type 1 isolates showed lack of association between their anatomic sites of infection and genotypic/sub genotypic classification.

BACKGROUND: HSV-1 genome is a mosaic of recombinants. Clinical Herpes simplex virus -1 (HSV1) isolates were already genotyped as A, B and C types based on nucleotide variations at Unique Short (US) 4 (gG) and US 7 (gI) regions through phylogeny. Analysis of Glycoprotein C (gC) exon present on the Unique Long (UL) region had also revealed the existence of different genotypes. Glycoprotein C is mainly involved in initial viral attachment to heparan sulphate on host cell surface facilitating the virus's binding and penetration into cell. As the amount of heparan sulphate on the host cell surface varies according to the cell type, it is plausible that different genotypes bind differentially to cell types. Hence, this study was framed to determine the existence of novel genotypes/sub genotypes in the US or UL regions which could associate with clinical entities. RESULTS: All the twenty five isolates analyzed in this study were of genotype A as per their gG gene sequences. In case of gI gene, 16 out of 25 were found to be type A and the remaining nine were type B putative intergenic recombinants. Intragenic recombinations were also encountered in both the US genes, with gG possessing novel subgenotypes, arbitrarily designated A1 and A2. The 9 type B isolates of gI genes also branched out into 2 clades due to genetic variations. Glycoprotein C of UL region had two distinct genotypic clades α and ß, whose topological distribution was significantly different from that of the US region. Neither the US nor UL regions, however, showed any preference among the genotypes to a specific anatomic site of infection. Even the non synonymous variations identified in the functional domain of gC, were not confined to a particular genotype/clinical entity. CONCLUSION: The analyses of the US and UL regions of the HSV-1 genome showed the existence of variegated genotypes in these two regions. In contrary to the documented literature, in which Asian strains were concluded as more conserved than European ones, our study showed the existence of a higher degree of variability among Indian strains. However, the identified novel genotypes and subgenotypes were not found associated with clinical entities.

Functional characterization of novel mutations in UL54 of ganciclovir resistant HCMV strain using structural analysis.

This study reports the probable impact of the coupled mutations observed in our clinical isolate of HCMV UL54 polymerase, through structural bioinformatics approaches. The reported variant was found to be resistant to Ganciclovir (GCV) as per the clinical records. The presence of Glutamine deletion at 639 (Glu639) and a mis sense mutation of Serine 655 Leucine (Ser655Leu) in UL54 were identified by DNA sequencing and were predicted to lie in the DNA polymerase type-II domain. Docking simulation studies of the phosphorylated forms of Ganciclovir (GCV), Cidofovir (CDV) and Foscarnet (PFA) with the reported mutants showed significant variation in terms of binding affinity and inhibitory constant (Ki) in comparison to wild type UL54. The findings of this study revealed that the observed coupled mutation could potentially induce allosteric effects in the binding pockets of UL54 and thereby alter the drug binding affinity. In specific, it was observed that this coupled mutation could confer changes in the binding affinity of GCV and PFA by altering the binding energies and inhibitory constants to -0.88Kcal/mol and 226.71mM, -5.81Kcal/mol and 54.83µM, respectively, in comparison to Wild Type. On the other hand, CDV showed increased susceptibility for the reported mutant with a binding energy of -6.16Kcal/mol and inhibitory constant of 30.47µM.