Case Series: Mixed Infectious Keratitis by Pythium insidiosum and Fungal Species.

SIGNIFICANCE: This case series is the first to illustrate mixed infection from Pythium sp. and fungal species in corneal ulcer. PURPOSE: This case series aimed to alert all toward the possibility of both Pythium sp. and fungal species infection in case of nonresponding corneal ulcer treated with either antifungals or antipythium drugs alone. Increased suspicion of mixed infection in case of nonresponding fungal/ Pythium keratitis may facilitate early and prompt management. CASE REPORTS: Six patients presented with signs of either fungal or Pythium keratitis. They underwent ophthalmological examinations, smear examinations, cultures, and polymerase chain reaction (PCR). Therapeutic penetrating keratoplasty was performed in cases where symptoms worsened after treatment with either antifungal or antipythium drugs. The half corneal button (HCB) was shared for histopathological and microbiological examinations. In the first case, smear examination from corneal scraping (CS) revealed Pythium -like filaments, which were confirmed with PCR; however, Aspergillus nidulans grew in culture. In the second case, iodine-potassium iodide (IKI) staining was positive for Pythium ; however, PCR was positive for both Pythium and fungus, which was further confirmed by DNA sequencing. In the third case, IKI staining and HCB were positive for Pythium ; however, PCR was positive for fungus, which was identified as Candida saitoana with DNA sequencing. In the fourth case, Pythium grew in the CS culture; however, Candida sp. grew in the HCB culture. In the fifth case, Cladosporium sp. grew in culture from CS; however, Pythium insidiosum grew from the anterior chamber exudate after therapeutic penetrating keratoplasty. In the sixth case, smear examination revealed septate fungal filaments, and Cladosporium sp. grew in culture; however, HCB on histopathological examination showed features of Pythium keratitis. CONCLUSIONS: In unresponsive cases of Pythium or fungal keratitis, diagnostic modalities such as IKI and PCR should be implemented as a routine practice, in addition to smears and cultures.

Repositioning the existing drugs for neuroinflammation: a fusion of computational approach and biological validation to counter the Parkinson's disease progression.

Parkinson's disease is caused by the deficiency of striatal dopamine and the accumulation of aggregated α-synuclein in the substantia nigra pars compacta (SNpc). Neuroinflammation associated with oxidative stress is a key factor contributing to the death of dopaminergic neurons in SNpc and advancement of Parkinson's disease. Two molecular targets, i.e., nuclear factor kappa-light-chain-enhancer (NF-kB) and α-synuclein play a substantial role in neuroinflammation progression. Therefore, the compounds targeting these neuroinflammatory targets hold a great potential to combat Parkinson's disease. Thereby, in this study, molecular docking and Connectivity Map (CMap) based gene expression profiling was utilized to reposition the approved drugs as neuroprotective agents for Parkinson's disease. With in silico screening, two drugs namely theophylline and propylthiouracil were selected for anti-neuroinflammatory activity evaluation in in vivo models of chronic neuroinflammation. The neuroinflammatory effect of the identified compounds was confirmed by quantifying the expression of three important neuroinflammatory mediators, i.e. IL-6, TNF-alpha, and IL-1 beta on brain tissue using ELISA assay. The ELISA experiment demonstrated that both compounds significantly decreased the expression of neuroinflammatory mediators, highlighting the compounds' potential in neuroinflammation management. Furthermore, the drug and disease interaction network of the two identified drugs and diseases (neuroinflammation and Parkinson's disease) suggested that the two drugs might interact with various targets namely adenosine receptors, Poly [ADP-ribose] polymerase-1, myeloperoxidase (MPO) and thyroid peroxidase through multiple pathways associated with neuroinflammation and Parkinson's disease. Computational studies suggest that a particular drug may be effective in managing Parkinson's disease associated with neuroinflammation. However, further research is needed to confirm this in biological experiments.

Microsporidia-induced stromal keratitis: a new cause of presumed immune stromal (interstitial) keratitis.

BACKGROUND AND OBJECTIVE: To describe the clinical features, diagnosis and management of immune stromal keratitis/interstitial keratitis (IK) associated with microsporidial epithelial keratitis. METHODS: Between October 2020 and January 2021, medical records of IK patients microbiologically proven as microsporidia from samples collected from corneal epithelium on smear examination, and/ or molecular analysis were reviewed. Demography, clinical profile and treatment were analysed. Real-time PCR (RT-PCR) for adenovirus (ADV), Epstein-Barr virus (EBV), herpes simplex virus (HSV) and varicella-zoster virus (VZV) was done. RESULTS: Twenty of 152 (13%) microbiologically proven cases of microsporidial keratitis were diagnosed as IK during the study period, the mean age and duration of symptoms were 35.7±11.4 years and 46.3±27.7 days, respectively. Half had predisposing risk factors, like trauma; and 30% had prior recurrences. One-fourth of patients were using antivirals on presentation. Characteristic presentations included disciform keratitis(n=12), incomplete/complete ring(n=5), and combination(n=3), along with variable subepithelial infiltrates (n=14). All cases had stromal oedema, with an intact epithelium and fine pigment dusting on endothelium. Corneal epithelial scrapings had scanty microsporidia spores in smears of 17/20 (85%), and pan-microsporidial DNA was identified in 14/20 (70%), with Vittaforma corneae by sequencing in 11/20 (55%). Other viruses detected were ADV (14,70%), VZV (2,10%), EBV (1,5%) and HSV (1,5%). Rapid resolution of inflammation and oedema within 2 weeks of starting steroids was seen in all cases. CONCLUSION: Microsporidia epithelial keratitis induced stromal inflammatory keratitis; is distinguished from microsporidial keratoconjunctivitis and stromal keratitis, by characteristic clinical features, and response to topical steroids.

Identification of novel MurA inhibitors using in silico approach, their validation and elucidation of mode of inhibition.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is an important enzyme involved in the first cytosolic step of bacterial cell wall synthesis. In this study a combination of ligand based and structure based in silico virtual screening methods were utilised for screening of more than 50,000 drug-like compounds from CSIR-IIIM in-house compound library in order to identify potent inhibitors of MurA. The identified hits were validated in vitro under various incubation conditions using Malachite green phosphate assay, and two potent hits viz 3772-9534 and D396-0012 were identified. Among these hits, compound 3772-9534 showed significant changes in the activity values in different assay conditions. The MD simulation study of 3772-9534 suggested a novel binding site in MurA enzyme, independent of the two-substrate binding sites. Binding of inhibitors at the allosteric site induces conformational changes in the enzyme, which leads to inhibition of enzymatic activity. Overall, the study offers new insight for targeting MurA, which may promote the discovery of novel MurA allosteric site inhibitors.

Diagnostic Performance and Clinical Utility of Conventional PCR Assay in Early Diagnosis of COVID-19 Associated Rhino-Orbito-Cerebral Mucormycosis.

Early diagnosis and treatment of rhino-orbital-cerebral mucormycosis (ROCM) are crucial. Potassium hydroxide with Calcofluorwhite (KOH + CFW) smears can demonstrate the fungal hyphae, but mixed infections caused by both mucorales and non-mucorales pose a diagnostic challenge. Polymerase chain reaction (PCR) can detect mixed infections and differentiate mucorales from non-mucorales. This study aimed to evaluate the utility of a single reaction PCR in the diagnosis of ROCM and the efficacy of nasal biopsy and endonasal swab in the detection of fungus. Sixty-six clinical samples were collected from 33 patients and were subjected to KOH + CFW smear, culture and PCR. PCR was performed using pan-fungal primers targeting the 28S large subunit rRNA gene, and the amplified products were further sequenced to identify the fungi. KOH + CFW smear, culture and PCR detected mucorales in 54.6%, 27.3% and 63.6% patients, respectively. PCR detected mixed infection in 51.5% patients compared to 9.1% by KOH + CFW smear. PCR detected fungus in 90% of nasal biopsies and 77.8% of endonasal swabs. Rhizopus spp. was the most common fungi identified in 43.2% of PCR-positive samples. PCR is effective in detecting mixed infection and in the diagnosis of ROCM. Nasal biopsies had better fungal detection rates than endonasal swabs.

Sequelae of microsporidial keratoconjunctivitis and its management.

Purpose: : To characterize the sequelae of microsporidia keratoconjunctivitis (MKC) and outline its management. Methods: Retrospective analysis of microbiologically proven MKC returned with persistent disease between January 2015 and December 2019 was done. Demographics, clinical features, management, and outcome were analyzed. Results: Sixteen patients (21 eyes) of 332 treated for MKC returned with the persisting disease. The mean age of 11 males (68.7%), and 5 females was 35.1 ± 12.2 years. Three-quarter of them did not have a known predisposing risk factor and one-quarter of them were referred for chronic conjunctivitis. Past medications included topical antivirals (n = 8) and topical corticosteroid (n = 6). Three predominant presentations were persistent (>3 weeks) superficial punctate keratitis (SPKs, n = 7), sub-epithelial infiltrates (SEIs, n = 13), and uveitis (n = 2). The lesions recurred in eight eyes (SPK and SEI 4 each) after a disease-free interval of 60.4 ± 40.6 days; there were 13 episodes of recurrence. Topical low potent corticosteroids (loteprednol/fluorometholone), and tacrolimus ointment 0.03% were used in 17 (80.9%) and 8 (38%) eyes, respectively, for a mean duration of 44.8 ± 31.6 and 226.8 ± 180.5 days, respectively. At follow-up, 172.3 ± 183.6 days, visual recovery was statistically significant in persistent eyes (BCVA 0.07 ± 0.07 logMAR; P < 0.00001) but, not in recurrent eyes (BCVA 0.16 ± 0.08 logMAR; P = 0.07). Five of 21 eyes were left with residual significant scar. Conclusion: The sequelae of microsporidial keratoconjunctivitis are not uncommon. Topical 0.03% tacrolimus ointment appeared to be an effective corticosteroid-sparing agent for the treatment of SEIs and prevention of recurrence.

Aurora Kinase B Expression, Its Regulation and Therapeutic Targeting in Human Retinoblastoma.

Purpose: Aurora kinase B (AURKB) plays a pivotal role in the regulation of mitosis and is gaining prominence as a therapeutic target in cancers; however, the role of AURKB in retinoblastoma (RB) has not been studied. The purpose of this study was to determine if AURKB plays a role in RB, how its expression is regulated, and whether it could be specifically targeted. Methods: The protein expression of AURKB was determined using immunohistochemistry in human RB patient specimens and immunoblotting in cell lines. Pharmacological inhibition and shRNA-mediated knockdown were used to understand the role of AURKB in cell viability, apoptosis, and cell cycle distribution. Cell viability in response to AURKB inhibition was also assessed in enucleated RB specimens. Immunoblotting was employed to determine the protein levels of phospho-histone H3, p53, p21, and MYCN. Chromatin immunoprecipitation-qPCR was performed to verify the binding of MYCN on the promoter region of AURKB. Results: The expression of AURKB was found to be markedly elevated in human RB tissues, and the overexpression significantly correlated with optic nerve and anterior chamber invasion. Targeting AURKB with small-molecule inhibitors and shRNAs resulted in reduced cell survival and increased apoptosis and cell cycle arrest at the G2/M phase. More importantly, primary RB specimens showed decreased cell viability in response to pharmacological AURKB inhibition. Additional studies have demonstrated that the MYCN oncogene regulates the expression of AURKB in RB. Conclusions: AURKB is overexpressed in RB, and targeting it could serve as a novel therapeutic strategy to restrict tumor cell growth.

Comparative profile of ocular surface microbiome in vernal keratoconjunctivitis patients and healthy subjects.

PURPOSE: To compare ocular surface microbiome and its antibiotic sensitivity in vernal keratoconjunctivitis (VKC) with normal ocular surface. METHODS: In this case-control study, thirty patients each with clinical diagnosis of VKC and age-matched controls with normal ocular surface were enrolled. Tear film samples were collected from each group and subjected to microbial evaluation with microscopy, conventional culture methods, and polymerase chain reaction (PCR). Microbial diversity and antibiotic sensitivity patterns were analyzed. RESULTS: Most patients (67%) belonged to severe grades (3 and 4) of VKC, and allergic history could be elicited in 20%. On culture, bacteria were isolated in 50% of VKC patients and 47% of control group. Staphylococcus species were identified in 70% VKC group and 57% control group. S. aureus growth was seen in 52% and 21% of VKC patients and controls, respectively. S. pneumoniae was isolated only in controls (29%) (p<0.05). Confluent colonies (≥10 colonies/µl) were seen in 70% of VKC patients and 14% of controls (p<0.05). Fluoroquinolone resistance was more among higher grades of VKC (50%) (p<0.01) and was observed in 46% of VKC patients and 23% of control group (p<0.01). Both groups were negative for HSV-1 DNA and fungal growth. CONCLUSION: Staphylococcus, the most common ocular surface flora, was predominant in VKC patients. Microbial analysis revealed similar microbial diversity in both groups. However, bacterial load was higher in VKC. Increased fluoroquinolone resistance was observed in VKC patients with more resistance among higher grades. Fungi and HSV-1 were not seen in VKC or normal ocular surface.

Antimicrobial peptides in human corneal tissue of patients with fungal keratitis.

BACKGROUND: Fungal keratitis (FK) is the leading cause of unilateral blindness in the developing world. Antimicrobial peptides (AMPs) have been shown to play an important role on human ocular surface (OS) during bacterial, viral and protozoan infections. In this study, our aim was to profile a spectrum of AMPs in corneal tissue from patients with FK during the active pase of infection and after healing. METHODS: OS samples were collected from patients at presentation by impression cytology and scraping. Corneal button specimens were collected from patients undergoing therapeutic penetrating keratoplasty for management of severe FK or healed keratitis. Gene expression of human beta-defensin (HBD)-1, -2, -3 and -9, S100A7, and LL-37 was determined by quantitative real-time PCR. RESULTS: Messenger RNA expression (mRNA) for all AMPs was shown to be significantly upregulated in FK samples. The levels of HBD-1 and -2 mRNA were found to be elevated in 18/20 FK samples. Whereas mRNA for HBD-3 and S100A7 was upregulated in 11/20 and HBD9 was increased in 15/20 FK samples. LL-37 mRNA showed moderate upregulation in 7/20 FK samples compared with controls. In healed scar samples, mRNA of all AMPs was found to be low and matching the levels in controls. CONCLUSION: AMP expression is a consistent feature of FK, but not all AMPs are equally expressed. HBD-1 and -2 are most consistently expressed and LL-37 the least, suggesting some specificity of AMP expression related to FK. These results will help to identify HBD sequence templates for designing FK-specific peptides to test for therapeutic potential.

Discovery of a New Donepezil-like Acetylcholinesterase Inhibitor for Targeting Alzheimer's Disease: Computational Studies with Biological Validation.

Alzheimer's disorder is one of the most common worldwide health problems, and its prevalence continues to increase, thereby straining the healthcare budgets of both developed and developing countries. So far, donepezil is the only Food and Drug Administration-approved dual-binding site inhibitor of acetylcholinesterase (AChE) that can amplify the cholinergic activity and also decrease Aß aggregation in Alzheimer patients. We report herein a new donepezil-like natural compound derivative (D1) as a convincing AChE inhibitor. The in silico studies suggests that D1 exhibits a dual-binding mode of action and interacts with both the catalytic anionic site and peripheral anionic site (PAS) of human AChE. The biological studies confirm the dual-binding site character of D1 and revealed that D1 not only enhances the acetylcholine levels but also reduces the accumulation of Aß plaques in Caenorhabditis elegans. In fact, 5 µM D1 was seen more potent in elevating the acetylcholine expression than 25 µM donepezil. While most of the non-cholinergic functions of donepezil, associated with the PAS of AChE, were gradually lost at higher concentrations, D1 was more functional at similar doses. Promisingly, D1 also exerted an agonistic effect on the α7 nicotinic acetylcholine receptor.

Combining ligand- and structure-based in silico methods for the identification of natural product-based inhibitors of Akt1.

The traditional method of drug discovery process has been surpassed by a rational approach where computer-aided drug designing plays a vital role in the identification of leads from large compound databases. Further, natural products have an important role in drug discovery as these have been the source of most active ingredients of medicines. Herein, in silico structure- and ligand-based approaches have been applied to screen in-house IIIM natural product repository for Akt1 (serine/threonine protein kinases) which is a well-known therapeutic target for cancer due to its overexpression and preventing the cells from undergoing apoptosis. Combined ligand-based and structure-based strategies were applied on to the existing library comprising of about 700 pure natural products, and the compounds identified from screening were biologically evaluated for Akt1 inhibition using Akt1 kinase activity assay. Fourteen promising compounds showed significant inhibition at 500 nM through in vitro screening, and from them, eight were new for Akt1 inhibition. Through the MD studies of Akt1 with the most active compound IN00145, it was inferred that Lys179, Glu191, Glu228, Ala230, Glu234 and Asp292 are the important amino acid residues which provide stability to the Akt1-IN00145 complex. Lead optimization studies were also performed around the actives to design better and selective inhibitors for Akt1. The results emphasized the successful application of virtual screening to identify new Akt1 inhibitor scaffolds that can be developed into a drug candidate in drug discovery programme.

High Prevalence of Biofilm-Forming MRSA in the Conjunctival Flora in Chronic Dacryocystitis.

OBJECTIVES: To report the microbiological spectrum of conjunctival flora and prevalence of biofilm-forming Methicillin-resistant Staphylococcus aureus (MRSA) in conjunctival flora in chronic dacryocystitis. DESIGN: Prospective, case-control study. METHODS: We included patients with unilateral chronic dacryocystitis, and their unaffected eyes as control. Microbiological profile and antibiotic susceptibility of the isolates was determined by standard microbiological procedures. S. aureus isolates were further evaluated for Methicillin resistance by Oxacillin resistance screening agar method and mecA polymerase chain reaction (PCR) and for biofilm synthesis by Congo red agar method, Microtitre plate (MTP) assay, and ica A and ica D PCR. RESULTS: We found 95 patients with unilateral chronic dacryocystitis. Aerobic Gram-positive isolates (74.2%, n = 72) were more than Gram-negative (25.7%, n = 25) or anaerobic isolates (20.5%, n = 25). S. aureus was most common (46.4%, n = 45), followed by Pseudomonas aeruginosa (10.3%, n = 10). Gram-positive isolates showed highest sensitivity to Linezolid (100%) and higher generation fluoroquinolones. Gram-negative isolates showed good sensitivity (>90%) to all tested antibiotics. S. aureus isolates showed MRSA prevalence as 93.5% and 96.9% by Oxacillin resistance screening agar method and mecA PCR, respectively. Biofilm formation was found in 71.8% MRSA isolates by MTP assay and 58.1% MRSA isolates were resistant to ≥3 classes of antibiotics. CONCLUSIONS: Gram-positive organisms, specifically S. aureus, are the major etiological agent in chronic dacryocystitis. There is high prevalence of MRSA in these isolates and concurrent biofilm formation.

Normalised quantitative polymerase chain reaction for diagnosis of tuberculosis-associated uveitis.

Polymerase chain reaction (PCR)-based diagnosis of tuberculosis-associated uveitis (TBU) in TB-endemic countries is challenging due to likelihood of latent mycobacterial infection in both immune and non-immune cells. In this study, we investigated normalised quantitative PCR (nqPCR) in ocular fluids (aqueous/vitreous) for diagnosis of TBU in a TB-endemic population. Mycobacterial copy numbers (mpb64 gene) were normalised to host genome copy numbers (RNAse P RNA component H1 [RPPH1] gene) in TBU (n = 16) and control (n = 13) samples (discovery cohort). The mpb64:RPPH1 ratios (normalised value) from each TBU and control sample were tested against the current reference standard i.e. clinically-diagnosed TBU, to generate Receiver Operating Characteristic (ROC) curves. The optimum cut-off value of mpb64:RPPH1 ratio (0.011) for diagnosing TBU was identified from the highest Youden index. This cut-off value was then tested in a different cohort of TBU and controls (validation cohort, 20 cases and 18 controls), where it yielded specificity, sensitivity and diagnostic accuracy of 94.4%, 85.0%, and 89.4% respectively. The above values for conventional quantitative PCR (≥1 copy of mpb64 per reaction) were 61.1%, 90.0%, and 74.3% respectively. Normalisation markedly improved the specificity and diagnostic accuracy of quantitative PCR for diagnosis of TBU.

ROLE OF EARLY VITRECTOMY IN THE TREATMENT OF FUNGAL ENDOPHTHALMITIS.

PURPOSE: To study whether immediate pars plana vitrectomy at the time of sampling vitreous for culture alter the visual and structural outcomes in fungal endophthalmitis. METHODS: Surgical results of 66 consecutive patients with culture-positive fungal endophthalmitis attending a tertiary eye care hospital in eastern India were analyzed. Based on the timing of vitrectomy, they were divided into 2 groups: Group 1-immediate vitrectomy (n = 31) and Group 2-diagnostic vitrectomy alone (n = 35) as the first-line intervention. The demographics, clinical features, and visual and structural outcomes were compared between the groups. RESULTS: The proportion of postoperative (54.83 vs. 51.42%), traumatic (29.03 vs. 34.28%), and endogenous (16.12 vs. 14.28%) endophthalmitis and clinical signs such as pain (54.83 vs. 60%), lid edema (48.38 vs. 48.57%), conjunctival congestion (87.09 vs. 94.28%), and corneal involvement (87.09 vs. 80%) in Groups 1 and 2, respectively, were comparable. Logarithm of the minimum angle of resolution vision improvement in Group 1 was significant (P = 0.0278) though there was no statistically significant difference in posttreatment vision between the groups (P = 0.322). Phthisis was seen in 25.8% in Group 1 and 25.7% in Group 2 (P = 0.993). Aspergillus species was the most common fungal pathogen isolated. Early vitrectomy plus intravitreal antifungals in a subset of clinically suspected fungal endophthalmitis resulted in statistically significant visual improvement over diagnostic vitrectomy plus intravitreal antifungals (P = 0.013). CONCLUSION: A strong clinical suspicion that translates into early vitrectomy plus intravitreal antifungal antibiotics leads to favorable visual and structural outcomes. A long wait till microbiological confirmation to institute antifungal therapy may result in poorer outcome.

A Novel Mutation in Helical Domain 2 of NOD2 in Sporadic Blau Syndrome.

We report a 12-year-old girl who presented with bilateral granulomatous anterior uveitis accompanied by boggy arthritis of knee and ankle joints, intermittent fever, and nodular skin rash. She was diagnosed with sporadic Blau syndrome (early-onset sarcoidosis) based on above clinical signs and presence of non-necrotising granuloma on iris biopsy. DNA sequencing revealed a previously unreported heterozygous mutation consisting of a G>A transition in exon 4 of the NOD2 gene. This resulted in a glutamic acid to lysine substitution in helical domain 2 of the nucleotide binding and oligomerization (NACHT) region, possibly reducing efficiency of auto-inhibition in NOD2 signaling. Interestingly, the ocular inflammation resolved completely following therapeutic vitrectomy in both eyes whereas the systemic symptoms of fever and arthritis continued to wax and wane while on treatment with oral methotrexate and corticosteroids.

Effect of Adjunctive Pars Plana Vitrectomy on Focal Posterior Segment Inflammation: A Case-Control Study in Tuberculosis-Associated Uveitis.

PURPOSE: Pars plana vitrectomy (PPV) is commonly performed for managing complications of uveitis but the anti-inflammatory potential of PPV has not been extensively investigated beyond aqueous/vitreous inflammation. We studied the effect of PPV on resolution of focal posterior segment lesions in tuberculosis-associated uveitis (TBU). DESIGN: Case control study. PARTICIPANTS: Patients with bilateral TBU and active retinal/choroidal lesions in both eyes, and who received PPV in one eye were included. Fellow eyes of same patients, matched for patient characteristics and systemic therapy, were designated as controls. METHODS: Study eyes received 3-port 23-guage PPV, involving removal of nearly the entire vitreous. Part of vitreous sample was used for quantitative polymerase chain reaction (qPCR) for Mycobacterium tuberculosis. Post-operatively, anti-TB and/or systemic corticosteroid therapy was initiated depending on level of clinical suspicion of tubercular etiology, degree of intraocular inflammation and qPCR results. Focal lesions were documented in preoperative and postoperative fundus diagrams. Clinical photographs were taken whenever adequate media clarity was present. MAIN OUTCOME MEASURES: Primary outcome measures were rate of clinical resolution of focal posterior segment lesions and improvement in best-corrected visual acuity (BCVA), at 1 month post-surgery. RESULTS: Thirty-six patients with bilateral posterior segment lesions consistent with TBU were included. Possible and probable TBU (depending on radiographic evidence of TB) were diagnosed in 28 (77.7%) and 4 (11.1%) patients respectively, whereas remaining 4 patients were diagnosed only on basis of qPCR results. Focal posterior segment lesions included retinal vasculitis (n = 27), multifocal-serpigenoid choroiditis (n = 5), multifocal choroiditis (n = 3) and focal choroiditis (n = 1). At one month postvitrectomy, 28 eyes (73.7%) showed complete resolution of focal posterior segment lesions compared to 7 non-vitrectomised eyes (19.4%), while improvement in BCVA was significantly more in study eyes (0.38 logarithm of the minimum angle of resolution [logMAR], P = 0.04), compared to controls (0.12 logMAR, P = 0.17). Time to resolution following vitrectomy was unaffected by duration of disease, pre-operative systemic steroids or grade of vitritis. At 3 months, complete resolution was noted in 29 of 30 study eyes (96.7%) and 25 of 30 control eyes (83.3%). CONCLUSIONS: PPV facilitates faster resolution of focal posterior segment inflammation and BCVA improvement in TBU.

Orbital tuberculosis: Clinicopathological correlation and diagnosis using PCR in formalin-fixed tissues.

This article describes the clinicopathological correlation and challenges encountered in the diagnosis of orbital tuberculosis (TB). Retrospective, interventional case series. A chart review of six patients who underwent biopsy for an orbital mass between January 2012 and December 2013 was performed. Institutional review board approval was obtained for the study. Clinical, radiological, and laboratory findings were documented, and response to antitubercular therapy (ATT) was noted. Age at presentation ranged from 18 to 64 years and duration of complaints varied from 2 weeks to 2 months. Pain, redness, and swelling with an orbital mass on computerized tomography (CT) were the common features. None of the patients was clinically suspected to have orbital TB at presentation. History of prior contact with a patient of TB and history of prior TB were present in one case each. Chest radiography (CXR) showed pulmonary consolidation in one case. Tuberculin skin test (TST) readings ranged from nonreactive to 23 mm. The presence of sputum acid fast bacilli (AFB) was tested in three cases and was negative. Polymerase chain reaction (PCR) for MPB64 gene was positive in all six cases. All cases responded to ATT. Clinical presentation of orbital TB can be variable and can simulate conditions like inflammatory disease and malignancy. Biopsy with histopathology plays a key role in diagnosis. Supportive laboratory investigations are necessary to clinch the diagnosis in cases where histopathology is suggestive of TB. In this regard, PCR for Mycobacterium tuberculosis (MTB) on tissue biopsy specimens may be a sensitive diagnostic tool.

Factors influencing polymerase chain reaction outcomes in patients with clinically suspected ocular tuberculosis.

BACKGROUND: Polymerase chain reaction (PCR) assay can be a useful method for definitive diagnosis in paucibacillary infections such as ocular tuberculosis (TB). In this study, we have evaluated factors affecting PCR outcomes in patients with clinically suspected ocular TB. Patients with clinically suspected ocular TB were investigated by PCR of aqueous or vitreous samples. Three control groups were also tested: group 1 included culture-proven non-tuberculous endophthalmitis, group 2 culture-negative non-tuberculous endophthalmitis, and group 3 patients undergoing surgery for uncomplicated cataract. PCR targeted one or more of following targets: IS6110, MPB64, and protein b genes of Mycobacterium tuberculosis complex. Multiple regression analysis (5% level of significance) was done to evaluate the associations between positive PCR outcome and laterality of disease, tuberculin skin test (TST)/interferon-gamma release assay (IGRA), chest radiography, and type of sample (aqueous or vitreous). The main outcome measures were positive PCR by one or more gene targets, and factors influencing positive PCR outcomes. RESULTS: All 114 samples were tested for MPB64, 110 for protein b, and 88 for IS6110. MPB64 was positive in 70.2% (n = 80) of tested samples, protein b in 40.0% (n = 44), and IS6110 in only 9.1% (n = 8). DNA sequencing of amplicons from four randomly chosen PCR reactions showed homology for M. tuberculosis complex. Of the 80 PCR-positive patients, 71 completed a full course of antitubercular therapy, of which 65 patients (91.5%) had complete resolution of inflammation at final follow-up. Among controls, 12.5% (3 out of 24) in group 1 and 18.7% (6 out of 32) in group 2 also tested positive by PCR. No PCR-positive outcome was observed in control group 3 (n = 25). Multiple regression analysis revealed significant association of positive PCR outcome with bilateral presentation, but not with a positive TST/IGRA, chest radiography, or type of sample (aqueous/vitreous) used. CONCLUSIONS: Careful selection of gene targets can yield high PCR positivity in clinically suspected ocular TB. Bilateral disease presentation but not any evidence of latent systemic TB influences PCR outcomes. False-positive results may be seen in ocular inflammation unrelated to ocular TB.

Development of a loop-mediated isothermal amplification assay targeting the mpb64 gene for diagnosis of intraocular tuberculosis.

A loop-mediated isothermal amplification (LAMP) assay targeting the mpb64 gene for the diagnosis of intraocular tuberculosis was highly specific (100%), sensitive (85.7%), rapid, and easy to perform. The LAMP assay can be an alternative to conventional PCR for the diagnosis of ocular tuberculosis in resource-limited settings.