In vivo confocal microscopy of trachoma in relation to normal tarsal conjunctiva.
ABSTRACT: To describe the in vivo confocal microscopy (IVCM) appearances of the tarsal conjunctiva in trachoma compared with the appearance of healthy conjunctiva and to develop grading systems for IVCM examination of the tarsal conjunctiva for use in future studies on trachoma and other conjunctival diseases.Prospective observational study.In vivo confocal microscopy examination was performed on 302 clinically normal adults, 16 clinically normal children, 750 adults with trachomatous conjunctival scarring, and 25 children with active trachoma.Clinical evaluation was performed with ×2.5 loupes, and IVCM examination of the upper tarsal conjunctiva was carried out with a Heidelberg Retina Tomograph 3 with the Rostock Cornea Module (Heidelberg Engineering GmbH, Dossenheim, Germany).In vivo confocal microscopy images were analyzed for cellular and tissue changes associated with trachomatous inflammation and scarring compared with healthy subjects.Trachomatous subjects with follicular and papillary inflammation had an increased inflammatory cellular infiltrate, including dendritiform cells, discrete follicular and papillary structures, and cystic lacunae suggestive of tissue edema. Trachomatous conjunctival scarring was seen with IVCM as organization of the subepithelial connective tissue into bands/sheets. Grading systems for inflammatory changes and scarring were developed, with the system for scarring showing good interobserver agreement with an intraclass coefficient of 0.88.In vivo confocal microscopy provides a powerful tool for examining the ocular surface. Numerous cellular and tissue changes were observed in subjects with trachoma, the first time IVCM has been applied to this disease. These changes both complement and add to previous histologic analyses. In vivo confocal microscopy promises to provide new insights into the pathogenesis of trachoma and other conjunctival diseases.
Project description:<h4>Objective</h4>To characterize the tissue and cellular changes found in trachomatous scarring (TS) and inflammation using in vivo confocal microscopy (IVCM).<h4>Design</h4>Two complimentary case-control studies.<h4>Participants</h4>The first study included 363 cases with TS (without trichiasis), of whom 328 had IVCM assessment, and 363 control subjects, of whom 319 had IVCM assessment. The second study included 34 cases with trachomatous trichiasis (TT), of whom 28 had IVCM assessment, and 33 control subjects, of whom 26 had IVCM assessment.<h4>Methods</h4>All participants were examined with ×2.5 loupes. The IVCM examination of the upper tarsal conjunctiva was carried out with a Heidelberg Retina Tomograph 3 with the Rostock Cornea Module (Heidelberg Engineering GmbH, Dossenheim, Germany).<h4>Main outcome measures</h4>The IVCM images were graded in a masked manner using a previously published grading system evaluating the inflammatory infiltrate density; the presence or absence of dendritiform cells (DCs), tissue edema, and papillae; and the level of subepithelial connective tissue organization.<h4>Results</h4>Subjects with clinical scarring had a characteristic appearance on IVCM of well-defined bands and sheets of scar tissue visible. Similar changes were also seen in some clinically normal subjects consistent with subclinical scarring. Scarred subjects had more DCs and an elevated inflammatory infiltrate, even after adjusting for other factors, including the level of clinical inflammation. Cellular activity was usually seen only in or just below the epithelium, rarely being seen deeper than 30 ?m from the surface. The presence of tissue edema was strongly associated with the level of clinical inflammation.<h4>Conclusions</h4>In vivo confocal microscopy can be quantitatively used to study inflammatory and scarring changes in the conjunctiva. Dendritic cells seem to be closely associated with the scarring process in trachoma and are likely to be an important target in antifibrotic therapies or the development of a chlamydial vaccine. The increased number of inflammatory cells seen in scarred subjects is consistent with the immunopathologic nature of the disease. The localization of cellular activity close to the conjunctival surface supports the view that the epithelium plays a central role in the pathogenesis of trachoma.<h4>Financial disclosure(s)</h4>The author(s) have no proprietary or commercial interest in any materials discussed in this article.
Project description:To compare in vivo confocal microscopy (IVCM) with the histopathological examination of tissue and cellular changes in normal and diseased conjunctiva.Participants underwent clinical examination and IVCM of the tarsal conjunctiva. A biopsy of the upper tarsal conjunctiva was collected and stained with tinctorial stains and by immunohistochemical staining for CD45 and CD83. Connective tissue scarring, inflammatory cell density and the presence of dendritiform cells were quantitatively assessed in a masked manner by both IVCM and histological assessments for comparative analysis.Thirty-four participants with severe trachomatous conjunctival scarring and 33 participants with healthy conjunctiva were recruited. The IVCM connective tissue scarring score was strongly associated with the histological grading of scarring (p<0.001). There was limited evidence of an association between the IVCM inflammatory cell infiltrate and the histological inflammatory cell grade (p=0.05). We did not find any evidence to support the hypothesis that dendritiform cells seen with IVCM are mature, conventional dendritic cells.The results show that IVCM can be used to robustly quantitate connective tissue scarring and also has a role in measuring the inflammatory cell infiltrate. The discordance between IVCM dendritiform cells and immunohistochemical dendritic cells may be a result of study limitations or may be because these dendritiform structures represent another cell type, such as fibroblasts, rather than dendritic cells.
Project description:Trachoma is the most common infectious cause of blindness and a major public health problem in many developing countries. It is caused by recurrent ocular infection with Chlamydia trachomatis in childhood, with conjunctival scarring seen later in life. The pathogenesis of trachomatous scarring, however, is poorly understood, and this study was carried out to investigate the immunofibrogenic correlates of trachomatous conjunctival scarring. A case-control study of 363 cases with conjunctival scarring and 363 control participants was conducted. Investigations included in vivo confocal microscopy (IVCM) assessment, quantitative real-time PCR gene expression, C. trachomatis detection, and nonchlamydial bacterial culture. Trachomatous scarring was found to be strongly associated with a proinflammatory, innate immune response with increased expression of psoriasin, interleukin-1?, tumor necrosis factor alpha, defensin-?4A, chemokine ligand 5, and serum amyloid A1. There was also differential expression of various modifiers of the extracellular matrix, including metalloproteinases 7, 9, 10, and 12, tissue inhibitor of matrix metalloproteinase 1, and secreted protein acidic cystein-rich-like 1. The expression of many of these genes was also significantly associated with the presence of nonchlamydial bacterial infection. These infections had a marked effect on conjunctival immune processes, including an increased inflammatory infiltrate and edema seen with IVCM. This study supports the possibility that the immunofibrogenic response in scarring trachoma is partly stimulated by nonchlamydial bacterial infection, which is characterized by the expression of innate factors.
Project description:Trichiasis is the sight-threatening consequence of conjunctival scarring in trachoma, the most common infectious cause of blindness worldwide. Trachomatous trichiasis is the result of multiple infections from childhood with Chlamydia trachomatis, which causes recurrent chronic inflammation in the tarsal conjunctiva. This produces conjunctival scarring, entropion, trichiasis, and ultimately blinding corneal opacification. The disease causes painful, usually irreversible sight loss. Over eight million people have trachomatous trichiasis, mostly those living in poor rural communities in 57 endemic countries. The global cost is estimated at US$ 5.3 billion. The WHO recommends surgery as part of the SAFE strategy for controlling the disease.We examine the principles of clinical management, treatment options, and the challenging issues of providing the quantity and quality of surgery that is needed in resource-poor settings.
Project description:Trachoma, caused by Chlamydia trachomatis, remains the world's leading infectious cause of blindness. Repeated ocular infection during childhood leads to scarring of the conjunctiva, in-turning of the eyelashes (trichiasis) and corneal opacity in later life. There is a growing body of evidence to suggest non-chlamydial bacteria are associated with clinical signs of trachoma, independent of C. trachomatis infection.We used deep sequencing of the V1-V3 region of the bacterial 16S rRNA gene to characterize the microbiome of the conjunctiva of 220 residents of The Gambia, 105 with healthy conjunctivae and 115 with clinical signs of trachoma in the absence of detectable C. trachomatis infection. Deep sequencing was carried out using the Roche-454 platform. Sequence data were processed and analyzed through a pipeline developed by the Human Microbiome Project.The microbiome of healthy participants was influenced by age and season of sample collection with increased richness and diversity seen in younger participants and in samples collected during the dry season. Decreased diversity and an increased abundance of Corynebacterium and Streptococcus were seen in participants with conjunctival scarring compared to normal controls. Abundance of Corynebacterium was higher still in adults with scarring and trichiasis compared to adults with scarring only.Our results indicate that changes in the conjunctival microbiome occur in trachomatous disease; whether these are a cause or a consequence is yet unknown.
Project description:Background: The low population-prevalence of trachomatous trichiasis and high prevalence of trachomatous inflammation-follicular (TF) provide contradictory estimates of the magnitude of the public health threat from trachoma in the Solomon Islands. Improved characterisation of the biology of trachoma in the region may support policy makers as they decide what interventions are required. Here, age-specific profiles of anti-Pgp3 antibodies and conjunctival scarring were examined to determine whether there is evidence of ongoing transmission and pathology from ocular Chlamydia trachomatis (Ct) infection. Methods: A total of 1511 individuals aged ?1 year were enrolled from randomly selected households in 13 villages in which >10% of children aged 1-9 years had TF prior to a single round of azithromycin mass drug administration undertaken six months previously. Blood was collected to be screened for antibodies to the Ct antigen Pgp3. Tarsal conjunctival photographs were collected for analysis of scarring severity. Results: Anti-Pgp3 seropositivity was 18% in 1-9 year olds, sharply increasing around the age of sexual debut to reach 69% in those over 25 years. Anti-Pgp3 seropositivity did not increase significantly between the ages of 1-9 years and was not associated with TF (p=0.581) or scarring in children (p=0.472). Conjunctival scars were visible in 13.1% of photographs. Mild (p<0.0001) but not severe (p=0.149) scars increased in prevalence with age. Conclusions: Neither conjunctival scars nor lymphoid follicles were associated with antibodies to Ct, suggesting that they are unlikely to be a direct result of ocular Ct infection . Clinical signs of trachoma were prevalent in this population but were not indicative of the underlying rates of Ct infection. The current World Health Organization guidelines for trachoma elimination indicated that this population should receive intervention with mass distribution of antibiotics, but the data presented here suggest that this may not have been appropriate.
Project description:INTRODUCTION:The phenotype and function of immune cells infiltrating the conjunctiva in scarring trachoma have yet to be fully characterized. We assessed tissue morphology and immunophenotype of cellular infiltrates found in trachomatous scarring compared to control participants. METHODOLOGY:Clinical assessments and conjunctival biopsy samples were obtained from 34 individuals with trachomatous scarring undergoing trichiasis surgery and 33 control subjects undergoing cataract or retinal detachment surgery. Biopsy samples were fixed in buffered formalin and embedded in paraffin wax. Hematoxylin and eosin (H&E) staining was performed for assessment of the inflammatory cell infiltrate. Immunohistochemical staining of single markers on individual sections was performed to identify cells expressing CD3 (T-cells), CD4 (helper T-cells), CD8 (suppressor/cytotoxic T-cells and Natural Killer, NK, cells), NCR1 (NK cells), CD20 (B-cells), CD45 (nucleated hematopoietic cells), CD56 (NK and T-cells), CD68 (macrophages/monocytes) and CD83 (mature dendritic cells). The degree of scarring was assessed histologically using cross-polarized light to visualize collagen fibres. PRINCIPLE FINDINGS:Scarring, regardless of clinical inflammation, was associated with increased inflammatory cell infiltrates on H&E and CD45 staining. Scarring was also associated with increased CD8+ and CD56+ cells, but not CD3+ cells, suggestive of a NK cell infiltrate. This was supported by the presence of NCR1+ cells. There was some increase in CD20+ cells, but no evidence for increased CD4+, CD68+ or CD83+ cells. Numerous CD45 negative cells were also seen in the population of infiltrating inflammatory cells in scarred conjunctiva. Disorganization of the normal collagen architecture was strongly associated with clinical scarring. CONCLUSIONS/SIGNIFICANCE:These data point to the infiltration of immune cells with a phenotype suggestive of NK cells in conjunctival trachomatous scarring. A large proportion of CD45 negative inflammatory cells were also present. Future work should seek to understand the stimuli leading to the recruitment of these cells and their role in progressive scarring.
Project description:Trachoma is a poorly understood immuno-fibrogenic disease process, initiated by Chlamydia trachomatis (Ct). Differences in conjunctival gene expression profiles between Tanzanians with trachomatous conjunctival scarring (with (TSI) and without (TS) inflammation) and controls (C) were investigated to identify relevant host responses. Tarsal conjunctival swab samples were collected for RNA isolation and Ct PCR. Transcriptome-wide microarray experiments were conducted on 41 samples (TSI 13, TS 15, C14). Cases had enrichment in proinflammatory cytokines and chemokines (IL1B, IL8, CXCL5, CCL20); Matrix metalloprotienases (MMP7, MMP9, MMP12); components of the cornified envelope / squamous metaplasia (SPRR2a, SPRR2F). Overall design: Case - control study. Conjunctival swab samples were collected from people with trachomatious conjunctival scarring with (TSI) and without (TS) clinically visable inflammation compared to normal controls (C). Total RNA extracted and analysed on the Illumina WG-6 platrom. Additional extensive qRT-PCR work done on a large set of case-control pairs.
Project description:Trachoma is a poorly understood immuno-fibrogenic disease process, initiated by Chlamydia trachomatis (Ct). Differences in conjunctival gene expression profiles between Tanzanians with trachomatous conjunctival scarring (with (TSI) and without (TS) inflammation) and controls (C) were investigated to identify relevant host responses. Tarsal conjunctival swab samples were collected for RNA isolation and Ct PCR. Transcriptome-wide microarray experiments were conducted on 41 samples (TSI 13, TS 15, C14). Cases had enrichment in proinflammatory cytokines and chemokines (IL1B, IL8, CXCL5, CCL20); Matrix metalloprotienases (MMP7, MMP9, MMP12); components of the cornified envelope / squamous metaplasia (SPRR2a, SPRR2F). Case - control study. Conjunctival swab samples were collected from people with trachomatious conjunctival scarring with (TSI) and without (TS) clinically visable inflammation compared to normal controls (C). Total RNA extracted and analysed on the Illumina WG-6 platrom. Additional extensive qRT-PCR work done on a large set of case-control pairs.
Project description:Trachoma surveillance is most commonly performed by direct observation, usually by non-ophthalmologists using the World Health Organization (WHO) simplified grading system. However, conjunctival photographs may offer several benefits over direct clinical observation, including the potential for greater inter-rater agreement. This study assesses whether inter-rater agreement of trachoma grading differs when trained graders review conjunctival photographs compared to when they perform conjunctival examinations in the field.Three trained trachoma graders each performed an independent examination of the everted right tarsal conjunctiva of 269 children aged 0-9 years, and then reviewed photographs of these same conjunctivae in a random order. For each eye, the grader documented the presence or absence of follicular trachoma (TF) and intense trachomatous inflammation (TI) according to the WHO simplified grading system.Inter-rater agreement for the grade of TF was significantly higher in the field (kappa coefficient, ?, 0.73, 95% confidence interval, CI 0.67-0.80) than by photographic review (??=?0.55, 95% CI 0.49-0.63; difference in ? between field grading and photo grading 0.18, 95% CI 0.09-0.26). When field and photographic grades were each assessed as the consensus grade from the three graders, agreement between in-field and photographic graders was high for TF (??=?0.75, 95% CI 0.68-0.84).In an area with hyperendemic trachoma, inter-rater agreement was lower for photographic assessment of trachoma than for in-field assessment. However, the trachoma grade reached by a consensus of photographic graders agreed well with the grade given by a consensus of in-field graders.