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© 2017 The Author(s). 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.

The putative vector of trachoma, Musca sorbens, prefers to lay its eggs on human faeces on the ground. This study sought to determine whether M. sorbens females were attracted to volatile odours from human faeces in preference to odours from the faeces of other animals, and to determine whether specific volatile semiochemicals mediate selection of the faeces. Traps baited with the faeces of humans and local domestic animals were used to catch flies at two trachoma-endemic locations in The Gambia and one in Ethiopia. At all locations, traps baited with faeces caught more female M. sorbens than control traps baited with soil, and human faeces was the most successful bait compared with soil (mean rate ratios 44.40, 61.40, 10.50 [P, Author summary Musca sorbens, also known as the Bazaar Fly, visits people’s faces to feed on ocular and nasal discharge. While feeding, M. sorbens can transmit Chlamydia trachomatis, the bacterium that causes the infectious eye disease trachoma. Around 1.9 million people worldwide are visually impaired or blind from this disease. Although it is believed that M. sorbens transmits trachoma, very few studies have looked at ways to control this fly. A large-scale trial has shown that control of fly populations with insecticide reduces active trachoma disease prevalence. Odour-baited traps for the suppression of disease vector populations are an attractive option as there is no widespread spraying of insecticide; however, highly attractive baits are critical to their success. Here, we demonstrate that the preference of these flies for breeding in human faeces is probably mediated by odour cues, and we isolate chemicals in the odour of human faeces that cause a response in the antennae of M. sorbens. These compounds may play a role in the specific attractiveness of human faeces to these flies, perhaps by being present in greater amounts or at favourable ratios. These may be developed into a chemical lure for odour-baited trapping to suppress M. sorbens populations.

BackgroundTrachoma, 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 hypothesised risk factors for development of active trachoma and conjunctival scarringMethodsOcular 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.FindingsIn children, active trachoma was not associated with significant changes in the ocular microbiome.Haemophilusenrichment 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 ofCorynebacterium. Increased abundance ofCorynebacteriumin scarring disease was associated with innate immune responses to the microbiota, dominated by altered mucin expression and increased matrix adhesion.InterpretationIn the absence of currentC. trachomatisinfection, changes in the ocular microbiome associate with antimicrobial and inflammatory responses that impair epithelial cell health. In scarring trachoma, expansion of ‘non-pathogenic’ bacteria such asCorynebacteriumand innate responses are coincident, warranting further investigation of this relationship. Comparisons between active and scarring trachoma supported the relative absence of type-1 interferon responses in scarring, whilst highlighting a common suppression of re-epithelialisation with altered epithelial and bacterial adhesion, likely contributing to development of scarring pathology.

Trachoma is caused byChlamydia 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

Urban areas are traditionally excluded from trachoma surveillance activities, but due to rapid expansion and population growth, the urban area of Brikama in The Gambia may be developing social problems that are known risk factors for trachoma. It is also a destination for many migrants who may be introducing active trachoma into the area. This study aimed to determine the prevalence and risk factors for follicular trachoma and trichiasis in Brikama.A community-based cross-sectional prevalence survey including 27 randomly selected households in 12 randomly selected enumeration areas (EAs) of Brikama. Selected households were offered eye examinations, and the severity of trachoma was graded according to WHO's simplified grading system. Risk factor data were collected from each household via a questionnaire.The overall prevalence of trachomatous inflammation-follicular (TF) in children aged 1-9 years was 3.8% (95% CI 2.5-5.6), and the overall prevalence of trichiasis in adults aged ≥15 years was 0.46% (95% CI 0.17-1.14). EA prevalence of TF varied from 0% to 8.4%. The major risk factors for TF were dirty faces (P0.01, OR = 9.23, 95% CI 1.97-43.23), nasal discharge (P = 0.039, OR = 5.11, 95% CI 1.08-24.10) and residency in Brikama for1 year (P = 0.047, OR = 7.78, 95% CI 1.03-59.03).Follicular trachoma can be considered to have been eliminated as a public health problem in Brikama according to WHO criteria. However, as the prevalence in some EAs is5%, it may be prudent to include Brikama in surveillance programmes. Trichiasis remains a public health problem (0.1%), and active case finding needs to be undertaken.

Summary of previous Ct micro-array antigen identification. (DOCX 16Â kb)

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., Author Summary Trachoma is caused by the bacterium Chlamydia trachomatis (Ct). Individuals who have previously been infected with Ct carry specific antibodies in their blood. Recent studies have suggested that these antibodies may be a good way to estimate the intensity of transmission of this bacterium in a population. Among people who do have antibodies (seropositives) there is variation in the amount that is detectable in their blood. Some people have such low levels that differentiating them from those who don’t have antibodies (seronegatives) is challenging. We used a new test for Ct antibodies on blood specimens from three countries. Our test worked extremely well, giving reproducible results when we tested the same samples multiple times. We compared four different methods for setting the position of the threshold line between seronegatives and seropositives. The estimated transmission intensity in each country varied depending on the threshold method used, but two methods that used statistical modelling algorithms to define the two groups performed consistently across all three countries’ samples. We recommend that future studies should consider adopting the statistical modelling approaches, as they are objective tests that require no reference material and allow for standardisation between studies.

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.

All households in 32 villages were provided with improved pit latrines as part of a trachoma control programme in a rural part of The Gambia. Latrine provision was externally driven and was not in response to a request from the communities involved. Materials were provided for free and labour was paid for. To assess durability and acceptability we conducted a follow-up study 25-47 months after construction. Before the intervention only 32% of households in these villages had access to any type of latrine; at follow-up this had risen to 95%. On visual inspection 585/666 latrines (87.3%) were usable and 510 (87.2% of those usable) were currently used. During interviews 566/637 latrine owners (89%) said they were either happy or very happy with their latrines, and 620 (97.3%) reported that they would make a new latrine of some kind when the current one was full or unusable. We interpret these data to suggest that externally driven latrine provision, without additional health education, to an area with poor latrine coverage can result in high, sustainable levels of uptake and generate future demand for sanitation.

OBJECTIVES To assess whether the trachoma vector Musca sorbens was breeding in household latrines in a trachoma-endemic part of The Gambia. METHODS Longitudinal study of flies emerging from 16 sentinel household latrines selected at random from a list of all latrines present in four Gambian villages. Latrines were surveyed and fly traps were set over the drop hole for 24 h once per month for a year. RESULTS All the sentinel latrines were of the 'Gambian improved household latrine' design, which has a cement slab but is not ventilated or fly-proofed. The latrines were all in regular use by a family, mean number of users per latrine 14.8 (SD 8.0, range 2-29). Of 55 351 flies caught in 192 catches 54 130 (97.8%) were Chrysomya albiceps, 690 (1.2%) Musca domestica, 466 (0.8%) Chrysomya regalis and 65 (0.1%) M. sorbens. Of the M. sorbens caught 61 (93.8%) were female. CONCLUSIONS Gambian improved household pit latrines cannot be considered a source of the trachoma vector M. sorbens, and the promotion of pit latrines as a method to reduce M. sorbens is warranted. A large number of C. albiceps were caught emerging from the latrines, but this species is not considered to be of medical importance.

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., Author Summary Chlamydia trachomatis is a pathogen that causes sexually transmitted infections (STIs) and the blinding disease trachoma. Natural Killer (NK) cells are part of the host immune system's first line of defence against infection. NK cell functions are genetically encoded and differences between individuals mean that some people are better able to respond to infections than others. We found that in certain combinations, specific variants of the gene HLA-C (Human Leucocyte Antigen, C) and of a complex set of genes called the Killer-cell Immunoglobulin-like Receptors (KIR) were associated with a six-fold increase in the relative risk of scarring tissue damage resulting from ocular C. trachomatis infection (trachoma). This combination of genetic variants may reduce the host's ability to effectively resolve infections and result in a harmful immune response that ultimately leads to tissue damage and scarring. KIR+ NK cells are potential cellular mediators of the damaging immune response. Previous studies have identified that the same HLA-KIR genetic constellation that associates with trachoma is actually protective against infectious diseases such as malaria and tuberculosis. The high frequency of the trachoma-associated constellation in African populations may therefore be explained by the evolutionary benefits of protection from the complications of severe disease.

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., Author Summary Trachoma, which results from infection with a bacterium Chlamydia trachomatis(Ct), is a leading cause of preventable blindness in the world. One of the currently used control methods is mass drug administration (MDA) with azithromycin, which is initiated according to rates of follicular trachoma(TF) in children. This study was a clinical trial done to determine whether testing communities for Ct infection will prevent unnecessary rounds of MDA. This was done by allowing communities to stop treatment if their infection had been reduced below a threshold. The study compared the effects of one round of mass treatment to three and found that there was no difference in either follicular trachoma or infection rates after three years. One round of treatment reduced TF to a low level. Tests for infection could be used to decide when to start or discontinue MDA and to prevent unnecessary treatment rounds in settings like The Gambia.

Summary Background Community-wide mass antibiotic treatment is a central component of trachoma control. The optimum frequency and duration of treatment are unknown. We measured the effect of mass treatment on the conjunctival burden of Chlamydia trachomatis in a Gambian community with low to medium trachoma prevalence and investigated the rate, route, and determinants of re-emergent infection. Methods 14 trachoma-endemic villages in rural Gambia were examined and conjunctival swabs obtained at baseline, 2, 6, 12, and 17 months. Mass antibiotic treatment with azithromycin was given to the community at baseline. C trachomatis was detected by qualitative PCR and individual infection load then estimated by real-time quantitative PCR. Findings C trachomatis was detected in 95 (7%) of 1319 individuals at baseline. Treatment coverage was 83% of the population (1328 of 1595 people). The effect of mass treatment was heterogeneous. In 12 villages all baseline infections (34 [3%] of 1062 individuals) resolved, and prevalence (three [0·3%]) and infection load remained low throughout the study. Two villages (baseline infection: 61 [24%] of 257 individuals) had increased infection 2 months after treatment (74 [30%]), after extensive contact with other untreated communities. Subsequently, this value reduced to less than half of that before treatment (25 [11%]). Interpretation Mass antibiotic treatment generally results in effective, longlasting control of C trachomatis in this environment. For low prevalence regions, one treatment episode might be sufficient. Infection can be reintroduced through contact with untreated populations. Communities need to be monitored for treatment failure and control measures implemented over wide geographical areas.

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, Author Summary Trachoma is a debilitating disease that affects 40 million people worldwide. It can cause progressive fibrosis of the upper eyelid and blindness, yet the mechanism is poorly understood. We have investigated the expression of short sequences of genetic material (microRNA) that regulate gene expression. We screened for the expression of 754 microRNA sequences (miR) in genetic material isolated from conjunctival swab samples from individuals in trachoma-endemic communities in The Gambia. This sample included healthy controls, individuals with trachomatous scarring and individuals with trachomatous scarring in the presence of clinically significant inflammation. We found 82 miR that were differentially expressed. Computer simulations predict that these miR regulate genes in epithelial cell differentiation, inflammation and fibrosis pathways, all of which are involved in the scarring process. We then validated the expression of seven of these differentially expressed miR in a second larger biological sample set from The Gambia. We confirmed that miR-147b and miR-1285 have increased expression in individuals with trachomatous scarring in the presence of clinically significant inflammation. Further investigation into the functions of these miR will aid our understanding of this disease and present opportunities to develop treatments for ocular fibrotic diseases.

Trachoma is the leading infectious cause of blindness. Conjunctival Chlamydia trachomatis infection causes scarring, entropion, trichiasis, and blinding corneal opacification. Worldwide, there are 8 million people with trichiasis. Although trichiasis surgery can reduce the risk of blindness, retrospective data suggest that long-term recurrence rates may be high. A 4-year prospective investigation of recurrent trichiasis was conducted in The Gambia.Patients with trichiasis were examined at baseline, 6 months, 1 year, and 4 years after posterior lamellar tarsal rotation surgery. Conjunctival swabs for bacteriology and PCR for C. trachomatis were collected at baseline, 6 months, and 1 year.Three hundred fifty-six Gambian patients were enrolled at baseline and 266 were reassessed at 4 years (94% of surviving patients). The recurrence rates were 32%, 40%, and 41% at 6 months, 1 year, and 4 years, respectively. At 4 years, 30% of patients had bilateral trichiasis and 21% had bilateral corneal opacity. Recurrence was associated with severe conjunctival inflammation and severe trichiasis (10 lashes) at baseline.Trichiasis recurrence rates were high, and most cases recurred within 6 months of surgery. The results suggest that there are important aspects of surgical technique and quality that should to be addressed. Persistent inflammation is strongly associated with recurrence at 4 years.

BACKGROUND: Trachoma is the leading infectious cause of blindness. Routes of transmission remain unclear. In this study, the relationship between Chlamydia trachomatis Amplicor-positive nasal discharge and Amplicor-positive ocular swabs was investigated (Amplicor; Roche, Indianapolis, IN). METHODS: A longitudinal study was conducted in Tanzania and The Gambia. Eyes were graded for active trachoma; ocular swabs were taken to test for C. trachomatis. Children with visible nasal discharge had swabs taken of this material. Participants were offered systemic antibiotics. Two months after treatment, participants were re-examined. RESULTS: Of the 1128 children participating, 188 (17%) had nasal discharge. Among 188 children with nasal discharge, 64 (34%) nasal swabs were PCR positive. There was a strong correlation between active disease/ocular chlamydial positivity and positive nasal discharge. Children with Amplicor-positive ocular swabs were 9.9 times more likely to have Amplicor-positive nasal discharge than were children without ocular positivity (95% CI: 4.34 – 22.53). Two months after treatment, 16% had an Amplicor-positive ocular swab. Children with positive nasal discharge at baseline were 5.2 times more likely to have an Amplicor-positive ocular swab at 2 months than were children without Amplicor-positive nasal discharge at baseline (95% CI: 1.54 – 17.23), after adjusting for baseline ocular positivity, gender, and study site. CONCLUSIONS: Nasal discharge may provide a source of reinfection with C. trachomatis, after antibiotic treatment for trachoma, either through transfer of secretions from nose to eye or from nasal secretions transferred to bed sheets or dirty clothes and back to the eye; alternatively, nasal discharge may be an indicator of severe persistent ocular chlamydial infection that is not cleared with a single dose of antibiotics.