Krupple-like factor 4 (KLF4) methylation signature in host cell in active viral keratitis with epithelial manifestation.

HSV1 presents as epithelial or stromal keratitis or keratouveitis and can lead to sight-threatening complications. KLF4, a critical transcription factor, and regulator of cell growth and differentiation, is essential in corneal epithelium stratification and homeostasis. Here, we want to understand the epigenetic modification specifically the methylation status of KLF4 in epithelium samples of HSV1 keratitis patients. After obtaining consent, epithelial scrapes were collected from 7 patients with clinically diagnosed HSV1 keratitis and 7 control samples (patients undergoing photorefractive keratectomy). Genomic DNA was isolated from the collected samples using the Qiagen DNeasy Kit. Subsequently, bisulfite modification was performed. The bisulphite-modified DNA was then subjected to PCR amplification using specific primers designed to target the KLF4, ACTB gene region, allowing for the amplification of methylated and unmethylated DNA sequences. The amplified DNA products were separated and visualized on a 3% agarose gel. KLF4 hypermethylation was found in 6 out of 7 (85.71%) eyes with viral keratitis, while 1 eye showed hypomethylation compared to PRK samples. Out of these 6, there were 2 each of epithelial dendritic keratitis, epithelial geographical keratitis, and neurotrophic keratitis. The patient with hypomethylated KLF4 had a recurrent case of HSV1 keratitis with multiple dendrites and associated vesicular lesions of the lip along with a history of fever. KLF4 hypermethylation in most viral keratitis cases indicated the under functioning of KLF4 and could indicate a potential association between KLF4 hypermethylation and the development or progression of HSV1 keratitis.

Immune activation markers in individuals with HIV-1 disease and their correlation with HIV-1 RNA levels in individuals on antiretroviral therapy.

BACKGROUND: Currently CD4+ T lymphocyte counts and HIV-1 RNA levels are being utilized to predict outcome of human immunodeficiency virus (HIV) disease. Recently, the role of immune activation in HIV disease progression and response to treatment is being investigated. This study focused on the expression of CD38 and HLA-DR on lymphocyte subsets in various groups of HIV-infected individuals and to determine their association with HIV-1 disease progression. METHODS: Ninety-eight cases of patients with HIV/AIDS in different disease stages and twenty-four healthy HIV-negative individuals were included in the cross-sectional study. Their immune function and abnormal immune activation markers (CD38 & HLA-DR) were detected using a flowcytometer, and HIV-1 RNA levels in individuals receiving antiretroviral drugs were estimated. RESULTS: The immune activation marker levels were significantly different between patients with different disease stages (P < 0.001). A significant negative correlation was observed between peripheral blood CD4+ T cell counts and immune activation markers. Also, a significant positive correlation was observed between HIV-1 RNA levels and CD38+CD8+ T lymphocyte. CONCLUSION: Immune activation markers (CD38 & HLA-DR) increase with disease progression. CD38+ on CD8+ T lymphocyte correlates well with HIV1 RNA levels in individuals failing on antiretroviral therapy.

In vitro Assay to Examine the Function of Tears on Corneal Epithelial Cells.

Tears contain numerous secreted factors, enzymes, and proteins that help in maintaining the homeostatic condition of the eye and also protect it from the external environment. However, alterations to these enzymes and/or proteins during pathologies such as mechanical injury and viral or fungal infections can disrupt the normal ocular homeostasis, further contributing to disease development. Several tear film components have a significant role in curbing disease progression and promoting corneal regeneration. Additionally, several factors related to disease progression are secreted into the tear film, thereby serving as a valuable reservoir of biomarkers. Tears are readily available and can be collected via non-invasive techniques or simply from contact lenses. Tears can thus serve as a valuable and easy source for studying disease-specific biomarkers. Significant advancements have been made in recent years in the field of tear film proteomics, lipidomics, and transcriptomics to allow a better understanding of how tears can be utilized to gain insight into the etiology of diseases. These advancements have enabled us to study the pathophysiology of various disease states using tear samples. However, the mechanisms by which tears help to maintain corneal homeostasis and how they are able to form the first line of defense against pathogens remain poorly understood and warrant detailed in vitro studies. Herein, we have developed an in vitro assay to characterize the functional importance of patient isolated tears and their components on corneal epithelial cells. This novel approach closely mimics real physiological conditions and could help the researchers gain insight into the underlying mechanisms of ocular pathologies and develop new treatments. Key features • This method provides a new technique for analyzing the effect of tear components on human corneal epithelial cells. • The components of the tears that are altered in response to diseases can be used as a biomarker for detecting ocular complications. • This procedure can be further employed as an in vitro model for assessing the efficacy of drugs and discover potential therapeutic interventions.

Prolonged Inflammation and Infectious Changes in the Corneal Epithelium Are Associated with Persistent Epithelial Defect (PED).

Purpose: Failure of rapid re-epithelialization within 10-14 days after corneal injury, even with standard supportive treatment, is referred to as persistent corneal epithelial (CE) defect (PED). Though an array of genes regulates reepithelization, their mechanisms are poorly understood. We sought to understand the network of genes driving the re-epithelialization in PED. Method: After obtaining informed consent, patients underwent an ophthalmic examination. Epithelial scrapes and tears samples of six PED patients and six individuals (control) undergoing photorefractive keratectomy (PRK) were collected. RNA isolation and quantification were performed using either the epithelial scrape taken from PED patients or from HCLE cells treated with control tears or tears of PED patients. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of a few important genes in CE homeostasis, inflammation, and cell-cell communication, viz., Kruppel-like factor 4 (KLF4), GPX4, IL6, TNFα, STING, IL8, desmoglein, and E-cadherin, among others. Their expressions were normalized with their respective housekeeping genes and fold changes were recorded. KLF4 localization and MMPs activity was carried out via immunofluorescence and zymography, respectively. Results: KLF4, a transcription factor important for CE homeostasis, was upregulated in tears-treated HCLE cells and downregulated in PED patients compared to the healthy PRK group. Cell-cell communication genes were also upregulated in tears-treated cells, whereas they were downregulated in the PED tissue group. Genes involved in proinflammation (IL6, 282-fold; TNFα, 43-fold; IL8, 4.2-fold) were highly upregulated in both conditions. MMP9 activity increased upon tears treatment. Conclusions: This study suggests that tears create an acute proinflammatory milieu driving the PED disease pathology, whereas the PED patients scrapes are an indicator of the chronic stage of the disease. Interferons, pro-inflammatory genes, and their pathways are involved in PED, which can be a potential target for inducing epithelialization of the cornea.

Epidemiological, clinical and mycological characteristics of pityriasis versicolor: Results of a study from a teaching hospital in rural part of Northern India.

Background and Objectives: Pityriasis versicolor is a common fungal infection of the skin which leads to the formation of scaly and discoloured small lesions on skin. The main objective of this study is to describe clinical and mycological characteristics and the predisposing factors in patients with pityriasis versicolor. Material and Methods: In this prospective, observational, hospital-based cross-sectional study, patients of all ages with clinically suspected lesions of pityriasis versicolor were included. After detailed history and thorough clinical examination, skin scrapings were examined with 10% potassium hydroxide (KOH) under light microscope. The scrapings were also subjected to culture examination. Results: A total of 113 patients [78 (69.0%) male; 35 (31%) female] were included in the study. A total of 87 (76.9%) patients were from rural area. Outdoor occupation and positive family history of pityriasis versicolor was present in 65 (57.5%) and 38 (33.6%) patients, respectively. Recurrent episodes were reported by 66 (60%) patients. Excessive sweating and oily skin were seen in 36 (31.8%) and 24 (21.1%) patients, respectively. History of occlusive clothing was present in 22 (19.4%) patients. Chest, back, and shoulders were affected in 36 (31.8%), 22 (19.4%), and 08 (07.0%) patients, respectively. Hypopigmented lesions were seen in 97 (85.8%) patients. Patches and macules were observed in 60 (53.1%) and 53 (46.9%) patients, respectively. A total of 27 (23.8%) patients reported mild prutitus. A total of 79 (69.9%) patients were KOH positive and culture negative, whereas 26 (23.0%) patients were KOH as well as culture positive. Eight (7.0%) patients were both KOH and culture negative. Conclusion: Pytiriasis versicolor is more common in young adults and males with the most common presentation of hypopigmentation lesions. In our study population, presentation with large patches was more common than macular lesions. Pruritus was more in patients with large patches than those with macules.

Prevalence pattern of MRSA from a rural medical college of North India: A cause of concern.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) has become a global challenge. The shift of this organism form hospital settings to community setting and increasing resistance to non-ß-lactams antibiotics have further aggravated the crisis. This trend in MRSA has necessitated the knowledge and sensitization about this agent among physicians in rural and peripheral settings. MATERIAL AND METHODS: Present study was undertaken at Department of Microbiology at rural medical college of North India from January 2017 to December 2019 (3 years). All the clinical samples collected with aseptic precautions were processed as per standard protocol. All the Staphylococcus aureus isolates cultured were subjected to antimicrobial susceptibility testing as per CLSI guidelines 2019. Screening for MRSA was done by CLSI recommended methods, such as cefoxitin disc (30 µg), oxacillin disc (1 µg), and oxacillin screen agar as per CLSI recommendation. RESULTS: A steady increase in number of MRSA isolates was observed from year 2017 to 2019 with overall prevalence being 33.7%. Most MRSA isolates were obtained from pus samples. Cefoxitin disc diffusion method is a dependable detection method compared to oxacillin disc diffusion and oxacillin screen agar for identification of MRSA. CONCLUSION: The rising trend of MRSA impresses upon the acute need of stringent infection control practices namely strict compliance to hand hygiene, prevention of misuse and overuse of antibiotics and a continuous surveillance program for MRSA. Also sensitization about this agent among the primary health physician is the need of hour to implement the control measures and limit its spread in communities.

Hymenolepis diminuta infection in a young boy from rural part of Northern India.

Hymenolepis diminuta (H. diminuta) is primarily a parasite of rats and mice. Humans are infected by eating meal contaminated with these arthropods. This infection is not seen commonly in Indian population. We present here a case report of infection with H. diminuta in a young boy from a rural area of the North India.

Fonsecaea pedrosoi: a rare etiology in fungal keratitis.

Fungal corneal ulcer is common in India due to tropical climate and a large agrarian population that is at risk. Fonsecaea pedrosoi is the most common agent of chromoblastomycosis, a chronic localized fungal infection of the skin and subcutaneous tissues mainly involving lower extremities. We report a rare case of corneal chromoblastomycosis caused by F.pedrosoi, which was successfully treated with topical Amphotericin B followed by a long course of oral antifungal therapy. To the best of our knowledge, this is the first case of mycotic keratitis caused by F.pedrosoi, from the foothills of Himalayas.

Meningitis Due to Cryptococcus gattii in an Immunocompetent Patient.

The incidence of cryptococcal infection is high in developing countries such as India. Cryptococcus gattii, formerly known as Cryptococcus neoformans var gattii, is an encapsulated yeast that causes disease in both immunocompetent and immunosupressed individuals. The organism enters via respiratory tract and causes a spectrum of illness ranging from asymptomatic infection to severe illness, including pneumonia and disseminated infection involving multiple sites, including the central nervous system, eyes and skin. Cryptococcal meningitis is generally considered as rare in immunocompetent patients; therefore, specific treatment is not implemented until the organism is identified or a cryptococcal antigen is detected. We describe the case of a 30-years-old man without prior medical history who presented with meningitis and was treated successfully. This case illustrates the importance of considering infectious causes such as C.gattii in the differential diagnosis of meningitis, regardless of the patient's immune status.

Rhinocerebral mucormycosis in a patient with type 1 diabetes presenting as toothache: a case report from Himalayan region of India.

Mucormycosis is an angioinvasive infection caused by ubiquitous filamentous fungi of the order Mucorales. It is a rapidly progressive fatal infection mostly reported in susceptible individuals, such as those with poorly controlled diabetes or those with defects in phagocytic function. Rhinocerebral mucormycosis is the most common type of mucormycosis in diabetic patients. This case report describes a 17-year-old girl with type 1 diabetes mellitus presenting with rhinocerebral mucormycosis. The patient presented with a history of toothache and facial pain with oedema of left half of face, periorbital oedema and depressed conciousness. She had hyperglycaemia with diabetic ketoacidosis and rapidly developed hemiparesis progressing to quadriparesis and died within 3 days of admission. The current report emphasises the importance of having a high index of suspicion when dealing with patients with diabetes presenting with facial pain or cellulitis and prompt initiation of medical therapy along with surgical debridement for control of rhinocerebral mucormycosis.

Mycobacterium fortuitum Peritonitis in a Patient on Continuous Ambulatory Peritoneal Dialysis (CAPD): A Case Report.

Mycobacterium fortuitum, an environmental organism, is capable of producing a variety of clinical infections such as cutaneous infections, abscesses and nosocomial infections. Rarely, it has been a documented as a cause of peritonitis in patients receiving continuous ambulatory peritoneal dialysis (CAPD). Continuous Ambulatory Peritoneal dialysis (CAPD) is one of the treatment options which are used for patients with end-stage renal disease (ESRD). Although peritonitis rates have declined in parallel with advances in peritoneal dialysis (PD) technology, peritonitis remains a leading complication of CAPD and it is the major cause for transfer to other methods of dialysis. We are reporting a case of M. fortuitum peritonitis in a patient who was undergoing CAPD, which was successfully treated. This case emphasizes the importance of mycobacterial cultures in patients with CAPD-associated peritonitis, whose routine cultures may yield no organisms.