Differential frequency of NKG2C/KLRC2 deletion in distinct African populations and susceptibility to Trachoma: a new method for imputation of KLRC2 genotypes from SNP genotyping data.

NKG2C is an activating receptor that is preferentially expressed on natural killer (NK) cells. The gene encoding NKG2C (killer cell lectin-like receptor C2, KLRC2) is present at different copy numbers in the genomes of different individuals. Deletion at the NKG2C locus was investigated in a case-control study of 1522 individuals indigenous to East- and West-Africa and the association with the ocular Chlamydia trachomatis infection and its sequelae was explored. The frequency of homozygous KLRC2 deletion was 13.7 % in Gambians and 4.7 % in Tanzanians. A significantly higher frequency of the deletion allele was found in West-Africans from the Gambia and Guinea-Bissau (36.2 % p = 2.105 × 10(-8), 26.8 % p = 0.050; respectively) in comparison to East-African Tanzanians where the frequency of the deletion is comparable to other human populations (20.9 %). We found no evidence for an association between the numbers of KLRC2 gene copies and the clinical manifestations of trachoma (follicular trachoma or conjunctival scarring). A new method for imputation of KLRC2 genotypes from single nucleotide polymorphism (SNP) data in 2621 individuals from the Gambia further confirmed these results. Our data suggest that NKG2C does not play a major role in trachomatous disease. We found that the deletion allele is present at different frequencies in different populations but the reason behind these differences is currently not understood. The new method offers the potential to use SNP arrays from genome wide association studies to study the frequency of KLRC2 deletion in other populations and its association with other diseases.

A systems serology approach to the investigation of infection-induced antibody responses and protection in trachoma.

Background: Ocular infections with Chlamydia trachomatis serovars A-C cause the neglected tropical disease trachoma. As infection does not confer complete immunity, repeated infections are common, leading to long-term sequelae such as scarring and blindness. Here, we apply a systems serology approach to investigate whether systemic antibody features are associated with susceptibility to infection. Methods: Sera from children in five trachoma endemic villages in the Gambia were assayed for 23 antibody features: IgG responses towards two C. trachomatis antigens and three serovars [elementary bodies and major outer membrane protein (MOMP), serovars A-C], IgG responses towards five MOMP peptides (serovars A-C), neutralization, and antibody-dependent phagocytosis. Participants were considered resistant if they subsequently developed infection only when over 70% of other children in the same compound were infected. Results: The antibody features assayed were not associated with resistance to infection (false discovery rate < 0.05). Anti-MOMP SvA IgG and neutralization titer were higher in susceptible individuals (p < 0.05 before multiple testing adjustment). Classification using partial least squares performed only slightly better than chance in distinguishing between susceptible and resistant participants based on systemic antibody profile (specificity 71%, sensitivity 36%). Conclusions: Systemic infection-induced IgG and functional antibody responses do not appear to be protective against subsequent infection. Ocular responses, IgA, avidity, or cell-mediated responses may play a greater role in protective immunity than systemic IgG.

Longitudinal changes in tear cytokines and antimicrobial proteins in trachomatous disease.

BACKGROUND: Trachoma is a neglected tropical disease caused by ocular infection with Chlamydia trachomatis, where repeated infections and chronic inflammation can ultimately result in scarring, trichiasis and blindness. While scarring is thought to be mediated by a dysregulated immune response, the kinetics of cytokines and antimicrobial proteins in the tear film have not yet been characterised. METHODOLOGY: Pooled tears from a Gambian cohort and Tanzanian cohort were semi-quantitatively screened using a Proteome Profiler Array to identify cytokines differentially regulated in disease. Based on this screen and previous literature, ten cytokines (CXCL1, IP-10, IFN-γ, IL-1ß, IL-8, IL-10, IL-12 p40, IL-1RA, IL-1α and PDGF), lysozyme and lactoferrin were assayed in the Tanzanian cohort by multiplex cytokine assay and ELISA. Finally, CXCL1, IP-10, IL-8, lysozyme and lactoferrin were longitudinally profiled in the Gambian cohort by multiplex cytokine assay and ELISA. RESULTS: In the Tanzanian cohort, IL-8 was significantly increased in those with clinically inapparent infection (p = 0.0086). Lysozyme, IL-10 and chemokines CXCL1 and IL-8 were increased in scarring (p = 0.016, 0.046, 0.016, and 0.037). CXCL1, IP-10, IL-8, lysozyme and lactoferrin were longitudinally profiled over the course of infection in a Gambian cohort study, with evidence of an inflammatory response both before, during and after detectable infection. CXCL1, IL-8 and IP-10 were higher in the second infection episode relative to the first (p = 0.0012, 0.044, and 0.04). CONCLUSIONS: These findings suggest that the ocular immune system responds prior to and continues to respond after detectable C. trachomatis infection, possibly due to a positive feedback loop inducing immune activation. Levels of CXC chemokines in successive infection episodes were increased, which may offer an explanation as to why repeated infections are a risk factor for scarring.

Susceptibility to sequelae of human ocular chlamydial infection associated with allelic variation in IL10 cis-regulation.

Trachoma, an infectious disease of the conjunctiva caused by Chlamydia trachomatis, causes scarring and blindness in some infected individuals but not others. In an African community where trachoma is endemic, we have previously identified an IL10 haplotype that is associated with increased risk of scarring complications. Here we examine the hypothesis that the risk haplotype (H-RISK) affects levels of IL10 expression in the conjunctiva during active trachoma infection. To overcome potential genetic and environmental confounders we used the method of allele-specific quantification, which involved identifying subjects in the community who had active trachoma and were also heterozygous for the H-RISK. We find that there is allelic variation in cis-regulation of IL10 in the conjunctiva during active trachoma, with the H-RISK generating relatively more IL10 transcripts than other haplotypes in this population (average difference in IL10 allelic transcripts in the conjunctiva of heterozygous individuals infected with C. trachomatis of 23% (95% confidence interval: 14-32%, P < 0.0001). These findings provide a plausible functional explanation for the observed genetic association, and support the hypothesis that an excessive IL10 response to C. trachomatis infection is a risk factor for scarring and blindness.

Innate immunity in ocular Chlamydia trachomatis infection: contribution of IL8 and CSF2 gene variants to risk of trachomatous scarring in Gambians.

BACKGROUND: Trachoma, a chronic keratoconjunctivitis caused by Chlamydia trachomatis, is the world's commonest infectious cause of blindness. Blindness is due to progressive scarring of the conjunctiva (trachomatous scarring) leading to in-turning of eyelashes (trichiasis) and corneal opacification. We evaluated the contribution of genetic variation across the chemokine and cytokine clusters in chromosomes 4q and 5q31 respectively to risk of scarring trachoma and trichiasis in a large case-control association study in a Gambian population. METHODS: Linkage disequilibrium (LD) mapping was used to investigate risk effects across the 4q and 5q31 cytokine clusters in relation to the risk of scarring sequelae of ocular Ct infection. Disease association and epistatic effects were assessed in a population based study of 651 case-control pairs by conditional logistic regression (CLR) analyses. RESULTS: LD mapping suggested that genetic effects on risk within these regions mapped to the pro-inflammatory innate immune genes interleukin 8 (IL8) and granulocyte-macrophage colony stimulatory factor (CSF2) loci. The IL8-251 rare allele (IL8-251 TT) was associated with protection from scarring trachoma (OR = 0.29 p = 0.027). The intronic CSF2_27348 A allele in chromosome 5q31 was associated with dose dependent protection from trichiasis, with each copy of the allele reducing risk by 37% (p = 0.005). There was evidence of epistasis, with effects at IL8 and CSF2 loci interacting with those previously reported at the MMP9 locus, a gene acting downstream to IL8 and CSF2 in the inflammatory cascade. CONCLUSION: innate immune response SNP-haplotypes are linked to ocular Ct sequelae. This work illustrates the first example of epistatic effects of two genes on trachoma.

Conjunctival Microbiome-Host Responses Are Associated With Impaired Epithelial Cell Health in Both Early and Late Stages of Trachoma.

Background: Trachoma, a neglected tropical disease, is the leading infectious cause of blindness and visual impairment worldwide. Host responses to ocular chlamydial infection resulting in chronic inflammation and expansion of non-chlamydial bacteria are hypothesized risk factors for development of active trachoma and conjunctival scarring. Methods: Ocular swabs from trachoma endemic populations in The Gambia were selected from archived samples for 16S sequencing and host conjunctival gene expression. We recruited children with active trachoma and adults with conjunctival scarring, alongside corresponding matched controls. Findings: In children, active trachoma was not associated with significant changes in the ocular microbiome. Haemophilus enrichment was associated with antimicrobial responses but not linked to active trachoma. Adults with scarring trachoma had a reduced ocular bacterial diversity compared to controls, with increased relative abundance of Corynebacterium. Increased abundance of Corynebacterium in scarring disease was associated with innate immune responses to the microbiota, dominated by altered mucin expression and increased matrix adhesion. Interpretation: In the absence of current Chlamydia trachomatis infection, changes in the ocular microbiome associate with differential expression of antimicrobial and inflammatory genes that impair epithelial cell health. In scarring trachoma, expansion of non-pathogenic bacteria such as Corynebacterium and innate responses are coincident, warranting further investigation of this relationship. Comparisons between active and scarring trachoma supported the relative absence of type-2 interferon responses in scarring, whilst highlighting a common suppression of re-epithelialization with altered epithelial and bacterial adhesion, likely contributing to development of scarring pathology.

An outbreak of acute haemorrhagic conjunctivitis associated with coxsackievirus A24 variant in The Gambia, West Africa.

OBJECTIVE: An outbreak of acute haemorrhagic conjunctivitis occurred in The Gambia, West Africa in 2011. Affected individuals presented with conjunctival haemorrhages, swelling and ocular discharge. In an effort to identify a causative agent of the disease, ocular swabs were taken from patients during the acute and convalescent phases. Total RNA was extracted from all samples and reverse-transcriptase PCR performed using primers specific for all enteroviruses. Resulting amplicons were sequenced and data compared to known sequences using the BLAST algorithm. RESULTS: Forty-eight swabs were included in the analysis. Of these, 21 acute and 9 convalescent swabs (65% of the total) gave positive PCR results. Sequence analysis of the resulting amplicons indicated 99% sequence identity with coxsackievirus A24 variant identified during independent outbreaks of acute haemorrhagic conjunctivitis around the world and suggest the Gambian outbreak was due to this virus.

Profiling and validation of individual and patterns of Chlamydia trachomatis-specific antibody responses in trachomatous trichiasis.

BACKGROUND: Ocular Chlamydia trachomatis (Ct) infection causes trachoma, the leading infectious cause of blindness. A Ct D/UW3 proteome microarray and sera from Gambian adults with trachomatous trichiasis (TT) or healthy matched controls previously identified several novel antigens, which suggested differential recognition in adults with TT. METHODS: We re-analysed this serological microarray data using more robust microarray analysis techniques accounting for typical problems associated with highly dimensional data. We examined the Ct-specific antibody profile concerning the overall diversity of responses, antigen expression stage and cellular localisation of antigens. We tested differentially recognised antigens by further serological testing of the screened sera and used larger independent sample sets for validation. RESULTS: Antibody responses identified High-Performance on antigens expressed early and late in the Ct developmental cycle and those secreted or localised to the outer membrane. Eight antigens were preferentially recognised by scarred individuals and one antigen by healthy individuals. Three of these antigens, two associated with scarring (CT667 and CT706) and one healthy-associated (CT442), were not associated with the presence or absence of scarring following specific serological testing of the arrayed sera and sera from larger, independent case-control cohorts. CONCLUSIONS: This study identified focussed Ct-specific antibody profiles targeting proteins expressed during entry and exit from cells and localised to interact with the host. A small panel of antibody responses could discriminate between adults with and without TT in a trachoma-endemic community. Heterogenous responses in the independent validation of these antibody targets highlighted the need for large sample sizes, clearly defined clinical phenotypes and follow-up work.

Serology reflects a decline in the prevalence of trachoma in two regions of The Gambia.

Trachoma is caused by Chlamydia trachomatis (Ct). It is targeted for global elimination as a public health problem. In 2014, a population-based cross-sectional study was performed in two previously trachoma-endemic areas of The Gambia. Participants of all ages from Lower River Region (LRR) (N = 1028) and Upper River Region (URR) (N = 840) underwent examination for trachoma and had blood collected for detection of antibodies against the Ct antigen Pgp3, by ELISA. Overall, 30 (1.6%) individuals had active trachoma; the prevalence in children aged 1-9 years was 3.4% (25/742) with no statistically significant difference in prevalence between the regions. There was a significant difference in overall seroprevalence by region: 26.2% in LRR and 17.1% in URR (p < 0.0001). In children 1-9 years old, seroprevalence was 4.4% in LRR and 3.9% in URR. Reversible catalytic models using information on age-specific seroprevalence demonstrated a decrease in the transmission of Ct infection in both regions, possibly reflecting the impact of improved access to water, health and sanitation as well as mass drug administration campaigns. Serological testing for antibodies to Ct antigens is potentially useful for trachoma programmes, but consideration should be given to the co-endemicity of sexually transmitted Ct infections.

Genome-wide profiling of humoral immunity and pathogen genes under selection identifies immune evasion tactics of Chlamydia trachomatis during ocular infection.

The frequency and duration of Chlamydia trachomatis (Ct) ocular infections decrease with age, suggesting development of partial immunity. However, there is a lack of clear correlates of immunity to Ct infection in humans. We screened sera from a cohort of Gambian children followed for six-months against a Ct-proteome microarray. At genome sequence level, we detected signatures of selection from a population of ocular Ct isolates from Guinea-Bissau. Together these approaches allowed us to highlight the focus of humoral responses and hypothesise new modes of pathogen immune evasion. Children who were susceptible to frequent and/or prolonged Ct infection had a less focussed antibody response, including preferential recognition of forty-two antigens. There was evidence of positive and purifying selection across the genome, but little balancing selection. In contrast, most antigens that were associated with susceptibility were under neutral selection. These data suggest an evasion strategy in which Ct presents a large panel of irrelevant antigens to the immune system to block or misdirect protective responses. Development of a focused immune response, possibly induced through vaccination, may be an effective strategy to promote protection to Ct infection.

Defining Seropositivity Thresholds for Use in Trachoma Elimination Studies.

BACKGROUND: Efforts are underway to eliminate trachoma as a public health problem by 2020. Programmatic guidelines are based on clinical signs that correlate poorly with Chlamydia trachomatis (Ct) infection in post-treatment and low-endemicity settings. Age-specific seroprevalence of anti Ct Pgp3 antibodies has been proposed as an alternative indicator of the need for intervention. To standardise the use of these tools, it is necessary to develop an analytical approach that performs reproducibly both within and between studies. METHODOLOGY: Dried blood spots were collected in 2014 from children aged 1-9 years in Laos (n = 952) and Uganda (n = 2700) and from people aged 1-90 years in The Gambia (n = 1868). Anti-Pgp3 antibodies were detected by ELISA. A number of visual and statistical analytical approaches for defining serological status were compared. PRINCIPAL FINDINGS: Seroprevalence was estimated at 11.3% (Laos), 13.4% (Uganda) and 29.3% (The Gambia) by visual inspection of the inflection point. The expectation-maximisation algorithm estimated seroprevalence at 10.4% (Laos), 24.3% (Uganda) and 29.3% (The Gambia). Finite mixture model estimates were 15.6% (Laos), 17.1% (Uganda) and 26.2% (The Gambia). Receiver operating characteristic (ROC) curve analysis using a threshold calibrated against external reference specimens estimated the seroprevalence at 6.7% (Laos), 6.8% (Uganda) and 20.9% (The Gambia) when the threshold was set to optimise Youden's J index. The ROC curve analysis was found to estimate seroprevalence at lower levels than estimates based on thresholds established using internal reference data. Thresholds defined using internal reference threshold methods did not vary substantially between population samples. CONCLUSIONS: Internally calibrated approaches to threshold specification are reproducible and consistent and thus have advantages over methods that require external calibrators. We propose that future serological analyses in trachoma use a finite mixture model or expectation-maximisation algorithm as a means of setting the threshold for ELISA data. This will facilitate standardisation and harmonisation between studies and eliminate the need to establish and maintain a global calibration standard.

Conjunctival FOXP3 expression in trachoma: do regulatory T cells have a role in human ocular Chlamydia trachomatis infection?

BACKGROUND: Trachoma, caused by ocular infection with Chlamydia trachomatis, remains the leading infectious cause of blindness and in 2002 was responsible for 3.6% of total global blindness. Although transmission can be successfully interrupted using antibiotics and improvements in public and personal hygiene, the long-term success of the control programmes advocated by the World Health Organization are still uncertain. For the complete control and prevention of trachoma, a vaccine would be highly desirable. Currently there are no licensed vaccines for trachoma, and no human vaccine trials have been conducted since the 1960s. A barrier to new attempts to design and introduce a vaccine is the identification of immunologic correlates of protective immunity or immunopathology. We studied important correlates of the immune response in a trachoma-endemic population in order to improve our knowledge of this disease. This is essential for the successful development of a vaccine against both ocular and genital C. trachomatis infection. METHODS AND FINDINGS: We used quantitative real-time PCR for C. trachomatis 16S rRNA to identify conjunctival infection. The expression of IFN-gamma, IDO, IL-10, and FOXP3 mRNA transcripts was measured. We evaluated the role of immune effector and regulatory responses in the control of chlamydial infection and in the resolution of clinical signs of trachoma in endemic communities in Gambia. All host transcripts examined were detectable even in normal conjunctiva. The levels of these transcripts were increased, compared to normal uninfected conjunctiva, when infection was detected, with or without clinical disease signs. Interestingly, when clinical disease signs were present in the absence of infection, the expression of a regulatory T cell transcription factor, FOXP3, remained elevated. CONCLUSIONS: There is evidence of an increase in the magnitude of the local anti-chlamydial cytokine immune responses with age. This increase is coupled to a decline in the prevalence of infection and active trachoma, suggesting that effective adaptive immunity is acquired over a number of years. The anti-chlamydial and inflammatory immune response at the conjunctival surface, which may control chlamydial replication, is closely matched by counter inflammatory or regulatory IL-10 expression. Differences in the level of FOXP3 expression in the conjunctiva may indicate a role for regulatory T cells in the resolution of the conjunctival immune response, which is important in protection from immunopathology. However, the expression of cytokines that control chlamydial replication and those that regulate the conjunctival immune response is not simply juxtaposed; the interaction between the infection and the clinical disease process is therefore more complex.

A coding polymorphism in matrix metalloproteinase 9 reduces risk of scarring sequelae of ocular Chlamydia trachomatis infection.

BACKGROUND: Trachoma, an infectious disease of the conjunctiva caused by Chlamydia trachomatis, is an important global cause of blindness. A dysregulated extracellular matrix (ECM) proteolysis during the processes of tissue repair following infection and inflammation are thought to play a key role in the development of fibrotic sequelae of infection, which ultimately leads to blindness. Expression and activity of matrix metalloproteinase 9 (MMP-9), a major effector of ECM turnover, is up-regulated in the inflamed conjunctiva of trachoma subjects. Genetic variation within the MMP9 gene affects in vitro MMP9 expression levels, enzymatic activity and susceptibility to various inflammatory and fibrotic conditions. METHODS: We genotyped 651 case-control pairs from trachoma endemic villages in The Gambia for coding single nucleotide polymorphisms (SNPs) in the MMP9 gene using the high-throughput Sequenom system. Single marker and haplotype conditional logistic regression (CLR) analysis for disease association was performed. RESULTS: The Q279R mutation located in exon 6 of MMP9 was found to be associated with lower risk for severe disease sequelae of ocular Chlamydia trachomatis infection. This mutation, which leads to a nonsynonymous amino-acid change within the active site of the enzyme may reduce MMP-9-induced degradation of the structural components of the ECM during inflammatory episodes in trachoma and its associated fibrosis. CONCLUSION: This work supports the hypothesis that MMP-9 has a role in the pathogenesis of blinding trachoma.

Conjunctival fibrosis and the innate barriers to Chlamydia trachomatis intracellular infection: a genome wide association study.

Chlamydia trachomatis causes both trachoma and sexually transmitted infections. These diseases have similar pathology and potentially similar genetic predisposing factors. We aimed to identify polymorphisms and pathways associated with pathological sequelae of ocular Chlamydia trachomatis infections in The Gambia. We report a discovery phase genome-wide association study (GWAS) of scarring trachoma (1090 cases, 1531 controls) that identified 27 SNPs with strong, but not genome-wide significant, association with disease (5 × 10(-6) > P > 5 × 10(-8)). The most strongly associated SNP (rs111513399, P = 5.38 × 10(-7)) fell within a gene (PREX2) with homology to factors known to facilitate chlamydial entry to the host cell. Pathway analysis of GWAS data was significantly enriched for mitotic cell cycle processes (P = 0.001), the immune response (P = 0.00001) and for multiple cell surface receptor signalling pathways. New analyses of published transcriptome data sets from Gambia, Tanzania and Ethiopia also revealed that the same cell cycle and immune response pathways were enriched at the transcriptional level in various disease states. Although unconfirmed, the data suggest that genetic associations with chlamydial scarring disease may be focussed on processes relating to the immune response, the host cell cycle and cell surface receptor signalling.

The conjunctival microbiome in health and trachomatous disease: a case control study.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Our results indicate that changes in the conjunctival microbiome occur in trachomatous disease; whether these are a cause or a consequence is yet unknown.

Conjunctival scarring in trachoma is associated with the HLA-C ligand of KIR and is exacerbated by heterozygosity at KIR2DL2/KIR2DL3.

BACKGROUND: Chlamydia trachomatis is globally the predominant infectious cause of blindness and one of the most common bacterial causes of sexually transmitted infection. Infections of the conjunctiva cause the blinding disease trachoma, an immuno-pathological disease that is characterised by chronic conjunctival inflammation and fibrosis. The polymorphic Killer-cell Immunoglobulin-like Receptors (KIR) are found on Natural Killer cells and have co-evolved with the Human Leucocyte Antigen (HLA) class I system. Certain genetic constellations of KIR and HLA class I polymorphisms are associated with a number of diseases in which modulation of the innate responses to viral and intracellular bacterial pathogens is central. METHODOLOGY: A sample of 134 Gambian pedigrees selected to contain at least one individual with conjunctival scarring in the F1 generation was used. Individuals (n = 830) were genotyped for HLA class I and KIR gene families. Family Based Association Tests and Case Pseudo-control tests were used to extend tests for transmission disequilibrium to take full advantage of the family design, genetic model and phenotype. PRINCIPLE FINDINGS: We found that the odds of trachomatous scarring increased with the number of genome copies of HLA-C2 (C1/C2 OR = 2.29 BHP-value = 0.006; C2/C2 OR = 3.97 BHP-value = 0.0004) and further increased when both KIR2DL2 and KIR2DL3 (C2/C2 OR = 5.95 BHP-value = 0.006) were present. CONCLUSIONS: To explain the observations in the context of chlamydial infection and trachoma we propose a two-stage model of response and disease that balances the cytolytic response of KIR expressing NK cells with the ability to secrete interferon gamma, a combination that may cause pathology. The data presented indicate that HLA-C genotypes are important determinants of conjunctival scarring in trachoma and that KIR2DL2/KIR2DL3 heterozygosity further increases risk of conjunctival scarring in individuals carrying HLA-C2.

Conjunctival MicroRNA expression in inflammatory trachomatous scarring.

PURPOSE: Trachoma is a fibrotic disease of the conjunctiva initiated by Chlamydia trachomatis infection. This blinding disease affects over 40 million people worldwide yet the mechanisms underlying its pathogenesis remain poorly understood. We have investigated host microRNA (miR) expression in health (N) and disease (conjunctival scarring with (TSI) and without (TS) inflammation) to determine if these epigenetic differences are associated with pathology. METHODS: We collected two independent samples of human conjunctival swab specimens from individuals living in The Gambia (n = 63 & 194). miR was extracted, and we investigated the expression of 754 miR in the first sample of 63 specimens (23 N, 17 TS, 23 TSI) using Taqman qPCR array human miRNA genecards. Network and pathway analysis was performed on this dataset. Seven miR that were significantly differentially expressed between different phenotypic groups were then selected for validation by qPCR in the second sample of 194 specimens (93 N, 74 TS, 22 TSI). RESULTS: Array screening revealed differential expression of 82 miR between N, TS and TSI phenotypes (fold change >3, p<0.05). Predicted mRNA targets of these miR were enriched in pathways involved in fibrosis and epithelial cell differentiation. Two miR were confirmed as being differentially expressed upon validation by qPCR. miR-147b is significantly up-regulated in TSI versus N (fold change = 2.3, p = 0.03) and miR-1285 is up-regulated in TSI versus TS (fold change = 4.6, p = 0.005), which was consistent with the results of the qPCR array. CONCLUSIONS: miR-147b and miR-1285 are up-regulated in inflammatory trachomatous scarring. Further investigation of the function of these miR will aid our understanding of the pathogenesis of trachoma.

Mass treatment with azithromycin for trachoma: when is one round enough? Results from the PRET Trial in the Gambia.

BACKGROUND: The World Health Organization has recommended three rounds of mass drug administration (MDA) with antibiotics in districts where the prevalence of follicular trachoma (TF) is ≥10% in children aged 1-9 years, with treatment coverage of at least 80%. For districts at 5-10% TF prevalence it was recommended that TF be assessed in 1-9 year olds in each community within the district, with three rounds of MDA provided to any community where TF≥10%. Worldwide, over 40 million people live in districts whose TF prevalence is estimated to be between 5 and 10%. The best way to treat these districts, and the optimum role of testing for infection in deciding whether to initiate or discontinue MDA, are unknown. METHODS: In a community randomized trial with a factorial design, we randomly assigned 48 communities in four Gambian districts, in which the prevalence of trachoma was known or suspected to be above 10%, to receive annual mass treatment with expected coverage of 80-89% ("Standard"), or to receive an additional visit in an attempt to achieve coverage of 90% or more ("Enhanced"). The same 48 communities were randomised to receive mass treatment annually for three years ("3×"), or to have treatment discontinued if Chlamydia trachomatis (Ct) infection was not detected in a sample of children in the community after mass treatment (stopping rule("SR")). Primary outcomes were the prevalence of TF and of Ct infection in 0-5 year olds at 36 months. RESULTS: The baseline prevalence of TF and of Ct infection in the target communities was 6.5% and 0.8% respectively. At 36 months the prevalence of TF was 2.8%, and that of Ct infection was 0.5%. No differences were found between the arms in TF or Ct infection prevalence either at baseline (Standard-3×: TF 5.6%, Ct 0.7%; Standard-SR: TF 6.1%, Ct 0.2%; Enhanced-3×: TF 7.4%, Ct 0.9%; and Enhanced-SR: TF 6.2%, Ct 1.2%); or at 36 months (Standard-3×: TF 2.3%, Ct 1.0%; Standard-SR TF 2.5%, Ct 0.2%; Enhanced-3× TF 3.0%, Ct 0.2%; and Enhanced-SR TF 3.2%, Ct 0.7% ). The implementation of the stopping rule led to treatment stopping after one round of MDA in all communities in both SR arms. Mean treatment coverage of children aged 0-9 in communities randomised to standard treatment was 87.7% at baseline and 84.8% and 88.8% at one and two years, respectively. Mean coverage of children in communities randomized to enhanced treatment was 90.0% at baseline and 94.2% and 93.8% at one and two years, respectively. There was no evidence of any difference in TF or Ct prevalence at 36 months resulting from enhanced coverage or from one round of MDA compared to three. CONCLUSIONS: The Gambia is close to the elimination target for active trachoma. In districts prioritised for three MDA rounds, one round of MDA reduced active trachoma to low levels and Ct infection was not detectable in any community. There was no additional benefit to giving two further rounds of MDA. Programmes could save scarce resources by determining when to initiate or to discontinue MDA based on testing for Ct infection, and one round of MDA may be all that is necessary in some settings to reduce TF below the elimination threshold.

Systemic effector and regulatory immune responses to chlamydial antigens in trachomatous trichiasis.

Trachomatous trichiasis (TT) caused by repeated or chronic ocular infection with Chlamydia trachomatis is the result of a pro-fibrotic ocular immune response. At the conjunctiva, the increased expression of both inflammatory (IL1B, TNF) and regulatory cytokines (IL10) have been associated with adverse clinical outcomes. We measured in vitro immune responses of peripheral blood to a number of chlamydial antigens. Peripheral blood effector cells (CD4, CD69, IFNγ, IL-10) and regulatory cells (CD4, CD25, FOXP3, CTLA4/GITR) were readily stimulated by C. trachomatis antigens but neither the magnitude (frequency or stimulation index) or the breadth and amount of cytokines produced in vitro [IL-5, IL-10, IL-12 (p70), IL-13, IFNγ, and TNFα] were significantly different between TT cases and their non-diseased controls. Interestingly we observed that CD4+ T cells account for <50% of the IFNγ positive cells induced following stimulation. Further investigation in individuals selected from communities where exposure to ocular infection with C. trachomatis is endemic indicated that CD3-CD56+ (classical natural killer cells) were a major early source of IFNγ production in response to C. trachomatis elementary body stimulation and that the magnitude of this response increased with age. Future efforts to unravel the contribution of the adaptive immune response to conjunctival fibrosis should focus on the early events following infection and the interaction with innate immune mediated mechanisms of inflammation in the conjunctiva.

Pathway-focused arrays reveal increased matrix metalloproteinase-7 (matrilysin) transcription in trachomatous trichiasis.

PURPOSE: Several genes that are associated with protection from or susceptibility to trachomatous trichiasis (TT) have been identified through genetic association studies. Yet there have been few studies in which gene expression profiles were assessed in TT cases and disease-free controls. The purpose was to identify genes that are differentially expressed in the upper tarsal conjunctiva of subjects with TT. METHOD: Pathway-focused gene arrays were used to screen conjunctival RNA expression of 226 gene transcripts of interest. The screening was followed by validation of differentially expressed genes by qRT-PCR on an independent set of samples. Three different techniques were then used to test for quantitative differences in the recovered conjunctival protein fraction. RESULTS: Focused arrays identified a set of 13 differentially expressed genes. Validation by qRT-PCR confirmed differential expression in four of these genes (COL1A1, COL7A1, MMP7, and TLR6). Increased expression of MMP7 was the only consistent differentially regulated gene in the conjunctival samples of trichiasis subjects. MMP7 was present in isolated conjunctival proteins and in the tissue culture supernatants of peripheral blood lymphocytes after stimulation. CONCLUSIONS: There is an imbalance in extracellular matrix turnover with minimal contribution of adaptive immune responses at this stage of trichiasis. There was little evidence of broad differential expression in genes characteristic of polar responses of adaptive T cells or macrophages. The control of the MMP7 response and its activity appears significant in the fibrotic changes observed in TT.