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Background/aimsThe WHO recommends 3–5 years of annual mass azithromycin distribution with at least 80% treatment coverage to districts with active trachoma prevalence over 10% among children. Here, we assess the efficacy of expanding the coverage target to at least 90% for trachoma control in a mesoendemic region of Niger.MethodsTwenty-four communities were randomised to a single day of azithromycin distribution with a coverage target of 80% of the community or up to 4 days of treatment, aiming for greater than 90% coverage. Distributions were annual and individuals above 6 months of age were treated. Children under 5 years of age were monitored for ocular chlamydia infection and active trachoma.ResultsAt baseline, ocular chlamydia prevalence was 20.5% (95% CI 9.8% to 31.2%) in the standard coverage arm and 21.9% (95% CI 11.3% to 32.5%) in the enhanced coverage arm, which reduced to 4.6% (95% CI 0% to 9.5%, p=0.008) and 7.1% (95% CI 2.7% to 11.4%, p<0.001) at 36 months, respectively. There was no significant difference in 36-month ocular chlamydia prevalence between the two arms (p=0.21). There was no difference in the rate of decline in ocular chlamydia between the two arms in a repeated measures model (p=0.80).ConclusionsFor annual mass azithromycin distribution programme to an entire community, there may be no additional benefit of increasing antibiotic coverage above the WHO’s 80% target.Trial registration numberNCT00792922, post-results.

ABSTRACTThe Chlamydia trachomatisplasmid is a virulence factor. Plasmid copy number, C. trachomatisload and disease severity were assessed in a treatment-naive population where trachoma is hyperendemic. By using droplet digital PCR, plasmid copy number was found to be stable (median, 5.34 [range, 1 to 18]) and there were no associations with C. trachomatisload or disease severity.

ABSTRACTDroplet digital PCR (ddPCR) is an emulsion PCR process that performs absolute quantitation of nucleic acids. We developed a ddPCR assay for Chlamydia trachomatisinfections and found it to be accurate and precise. Using PCR mixtures containing plasmids engineered to include the PCR target sequences, we were able to quantify with a dynamic range between 0.07 and 3,160 targets/µl (r2= 0.9927) with >95% confidence. Using 1,509 clinical conjunctival swab samples from a population in which trachoma is endemic in Guinea Bissau, we evaluated the specificity and sensitivity of the quantitative ddPCR assay in diagnosing ocular C. trachomatisinfections by comparing the performances of ddPCR and the Roche Amplicor CT/NG test. We defined ddPCR tests as positive when we had =95% confidence in a nonzero estimate of target load. The sensitivity of ddPCR against Amplicor was 73.3% (95% confidence interval [CI], 67.9 to 78.7%), and specificity was 99.1% (95% CI, 98.6 to 99.6%). Negative and positive predictive values were 94.6% (95% CI, 93.4 to 95.8%) and 94.5% (95% CI, 91.3 to 97.7%), respectively. Based on Amplicor CT/NG testing, the estimated population prevalence of C. trachomatisocular infection was ~17.5%. Receiver-operator curve analysis was used to select critical cutoff values for use in clinical settings in which a balance between higher sensitivity and specificity is required. We concluded that ddPCR is an effective diagnostic technology suitable for both research and clinical use in diagnosing ocular C. trachomatisinfections.

To review recent clinical and epidemiological studies regarding the prevention, diagnosis, and treatment of trachoma.

ABSTRACTTrachoma 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 trachomatisin 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 vivoconfocal microscopy (IVCM) assessment, quantitative real-time PCR gene expression, C. trachomatisdetection, and nonchlamydial bacterial culture. Trachomatous scarring was found to be strongly associated with a proinflammatory, innate immune response with increased expression of psoriasin, interleukin-1β, tumor necrosis factor alpha, defensin-β4A, 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.

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-1β, tumor necrosis factor alpha, defensin-β4A, 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.

The immunological basis of scarring trachoma is not well understood. It is unclear whether it is driven primarily through cell-mediated adaptive or epithelial-cell-derived innate responses. The purpose of this study was to investigate the expression of the inflammatory and fibrogenic mediators which may be involved. We conducted a cross-sectional survey of children living in an untreated trachoma-endemic community in Tanzania. The children were examined for signs of trachoma, and swabs were collected for bacteriological culture and RNA and DNA isolation. Chlamydia trachomatis was detected by the Amplicor PCR test. The expression of the following genes was measured by quantitative reverse transcription-PCR (RT-PCR): S100A7, IL1B, IL17A, IL23A, CXCL5, CCL18, TLR2, NLRP3, KLRD1, CTGF, and MMP9. Four hundred seventy children under the age of 10 years were included. Follicular trachoma (TF) was detected in 65 children (14%), C. trachomatis was detected in 25 (5%), and bacterial pathogens were cultured in 161 (34%). TF was associated with significantly increased expression of S100A7, IL17A, CCL18, CXCL5, and CTGF. Expression was increased further in the presence of papillary inflammation. Nonchlamydial bacterial infection was associated with increased expression of IL17A, CXCL5, CCL18, and KLRD1. IL17A expression was associated with increased expression of S100A7, CXCL5, CCL18, KLRD1, and CTGF. These data are consistent with a role for IL-17A in orchestrating the proinflammatory response in trachoma. Its activity may be promoted either as part of the cell-mediated response or through innate pathways. It may drive a range of proinflammatory factors leading to excessive tissue damage and repair involving fibrosis.

ABSTRACTThe immunological basis of scarring trachoma is not well understood. It is unclear whether it is driven primarily through cell-mediated adaptive or epithelial-cell-derived innate responses. The purpose of this study was to investigate the expression of the inflammatory and fibrogenic mediators which may be involved. We conducted a cross-sectional survey of children living in an untreated trachoma-endemic community in Tanzania. The children were examined for signs of trachoma, and swabs were collected for bacteriological culture and RNA and DNA isolation. Chlamydia trachomatiswas detected by the Amplicor PCR test. The expression of the following genes was measured by quantitative reverse transcription-PCR (RT-PCR): S100A7, IL1B, IL17A, IL23A, CXCL5, CCL18, TLR2, NLRP3, KLRD1, CTGF, and MMP9. Four hundred seventy children under the age of 10 years were included. Follicular trachoma (TF) was detected in 65 children (14%), C. trachomatiswas detected in 25 (5%), and bacterial pathogens were cultured in 161 (34%). TF was associated with significantly increased expression of S100A7, IL17A, CCL18, CXCL5, and CTGF.Expression was increased further in the presence of papillary inflammation. Nonchlamydial bacterial infection was associated with increased expression of IL17A, CXCL5, CCL18, and KLRD1. IL17Aexpression was associated with increased expression of S100A7, CXCL5, CCL18, KLRD1, and CTGF.These data are consistent with a role for IL-17A in orchestrating the proinflammatory response in trachoma. Its activity may be promoted either as part of the cell-mediated response or through innate pathways. It may drive a range of proinflammatory factors leading to excessive tissue damage and repair involving fibrosis.

ABSTRACTTrachoma is the leading infectious cause of blindness and is endemic in 52 countries. There is a critical need to further our understanding of the host response during disease and infection, as millions of individuals are still at risk of developing blinding sequelae. Infection of the conjunctival epithelial cells by the causative bacterium, Chlamydia trachomatis, stimulates an acute host response. The main clinical feature is a follicular conjunctivitis that is incompletely defined at the tissue-specific gene expression and molecular levels. To explore the features of disease and the response to infection, we measured host gene expression in conjunctival samples from Gambian children with active trachoma and healthy controls. Genome-wide expression and transcription network analysis identified signatures characteristic of the expected infiltrating immune cell populations, such as neutrophils and T/B lymphocytes. The expression signatures were also significantly enriched for genes in pathways which regulate NK cell activation and cytotoxicity, antigen processing and presentation, chemokines, cytokines, and cytokine receptors. The data suggest that in addition to polymorph and adaptive cellular responses, NK cells may contribute to a significant component of the conjunctival inflammatory response to chlamydial infection.

Trachoma is the leading infectious cause of blindness and is endemic in 52 countries. There is a critical need to further our understanding of the host response during disease and infection, as millions of individuals are still at risk of developing blinding sequelae. Infection of the conjunctival epithelial cells by the causative bacterium, Chlamydia trachomatis, stimulates an acute host response. The main clinical feature is a follicular conjunctivitis that is incompletely defined at the tissue-specific gene expression and molecular levels. To explore the features of disease and the response to infection, we measured host gene expression in conjunctival samples from Gambian children with active trachoma and healthy controls. Genome-wide expression and transcription network analysis identified signatures characteristic of the expected infiltrating immune cell populations, such as neutrophils and T/B lymphocytes. The expression signatures were also significantly enriched for genes in pathways which regulate NK cell activation and cytotoxicity, antigen processing and presentation, chemokines, cytokines, and cytokine receptors. The data suggest that in addition to polymorph and adaptive cellular responses, NK cells may contribute to a significant component of the conjunctival inflammatory response to chlamydial infection.

Trachoma is a poorly understood immunofibrogenic disease process, initiated by Chlamydia trachomatis. Differences in conjunctival gene expression profiles between Ethiopians with trachomatous trichiasis (with [TTI] or without [TT] inflammation) and controls (C) were investigated to identify relevant host responses. Tarsal conjunctival swab samples were collected for RNA isolation and C. trachomatis PCR. Transcriptome-wide microarray experiments were conducted on 42 samples (TTI, n = 13; TT, n = 15; C, n =14). Specific results were confirmed by using multiplex quantitative reverse transcription-PCR for 16 mRNA targets in an independent collection of case-control samples: 386 case-control pairs (TTI, n = 244; TT, n = 142; C, n = 386). The gene expression profiles of cases were consistent with squamous metaplasia (keratins, SPRR), proinflammatory cytokine production (IL1β, CXCL5, and S100A7), and tissue remodeling (MMP7, MMP9, MMP12, and HAS3). There was no difference in the level of IFN between cases and controls. However, cases had increased INDO, NOS2A, and IL13RA2 and reduced IL13. C. trachomatis was detected in 1/772. Cases show evidence of ongoing inflammation and tissue remodeling, which were more marked where clinical inflammation was also present. Significantly, these processes appear to be active in the absence of current C. trachomatis infection. There was limited evidence of a TH1 response (INDO and NOS2A) and no association between a TH2 response and cases. The epithelium appears to be actively involved in late cicatricial stages of trachoma through the production of proinflammatory factors (IL1β, CXCL5, and S100A7). Longitudinal studies are needed to investigate which etiological factors and pathways are associated with progressive scarring and whether simply controlling chlamydial infection will halt progression in people with established cicatricial disease.

ABSTRACTTrachoma is a poorly understood immunofibrogenic disease process, initiated by Chlamydia trachomatis. Differences in conjunctival gene expression profiles between Ethiopians with trachomatous trichiasis (with [TTI] or without [TT] inflammation) and controls (C) were investigated to identify relevant host responses. Tarsal conjunctival swab samples were collected for RNA isolation and C. trachomatisPCR. Transcriptome-wide microarray experiments were conducted on 42 samples (TTI, n= 13; TT, n= 15; C, n=14). Specific results were confirmed by using multiplex quantitative reverse transcription-PCR for 16 mRNA targets in an independent collection of case-control samples: 386 case-control pairs (TTI, n= 244; TT, n= 142; C, n= 386). The gene expression profiles of cases were consistent with squamous metaplasia (keratins, SPRR), proinflammatory cytokine production (IL1β, CXCL5, and S100A7), and tissue remodeling (MMP7, MMP9, MMP12, and HAS3). There was no difference in the level of IFNγ between cases and controls. However, cases had increased INDO, NOS2A, and IL13RA2and reduced IL13. C. trachomatiswas detected in 1/772. Cases show evidence of ongoing inflammation and tissue remodeling, which were more marked where clinical inflammation was also present. Significantly, these processes appear to be active in the absence of current C. trachomatisinfection. There was limited evidence of a TH1 response (INDOand NOS2A) and no association between a TH2 response and cases. The epithelium appears to be actively involved in late cicatricial stages of trachoma through the production of proinflammatory factors (IL1β, CXCL5, and S100A7). Longitudinal studies are needed to investigate which etiological factors and pathways are associated with progressive scarring and whether simply controlling chlamydial infection will halt progression in people with established cicatricial disease.

Chlamydia-specific cytotoxic T lymphocytes are able to control model infections but may be implicated in disease pathogenesis. HLA-A2 peptide tetramers to Chlamydia trachomatis major outer membrane protein 258-266 (MOMP258-266) and MOMP260-268 were used to characterize HLA class I-restricted CD8+ T cells in Gambian children aged 4 to 15 years with clinical signs of active trachoma and/or infection with C. trachomatis. The frequencies of circulating HLA-A2 tetramer binding cells (TBC) were determined in whole blood samples by flow cytometric analysis. Initial screening of subjects with an anti-HLA-A2 antibody confirmed the presence of either HLA-A2 or HLA-A28. These were subsequently further divided by molecular subtyping. The C. trachomatis-specific HLA-A2 peptide tetramers were able to bind T cells with receptors from subjects which were restricted by either the HLA-A2 or the HLA-A28 restriction element. In this population, the median value of C. trachomatis-specific CD8+ T cells was 0.02%, with frequencies of up to 3.71% of CD8+ T cells reactive with a single tetramer in a minority of subjects. TBC were detected more often in subjects who were infected at the ocular surface, and their presence was associated with infection episodes of longer duration. Detection of C. trachomatis-specific TBC was not associated with the presence of disease or with the estimated load of ocular C. trachomatis infection at the time of sample collection. High frequencies of C. trachomatis-specific cells did not predict subsequent appearance or resolution of the clinical disease signs of active trachoma.

ABSTRACTChlamydia-specific cytotoxic T lymphocytes are able to control model infections but may be implicated in disease pathogenesis. HLA-A2 peptide tetramers to Chlamydia trachomatismajor outer membrane protein 258-266 (MOMP258-266) and MOMP260-268 were used to characterize HLA class I-restricted CD8+T cells in Gambian children aged 4 to 15 years with clinical signs of active trachoma and/or infection with C. trachomatis. The frequencies of circulating HLA-A2 tetramer binding cells (TBC) were determined in whole blood samples by flow cytometric analysis. Initial screening of subjects with an anti-HLA-A2 antibody confirmed the presence of either HLA-A2 or HLA-A28. These were subsequently further divided by molecular subtyping. The C. trachomatis-specific HLA-A2 peptide tetramers were able to bind T cells with receptors from subjects which were restricted by either the HLA-A2 or the HLA-A28 restriction element. In this population, the median value of C. trachomatis-specific CD8+T cells was 0.02%, with frequencies of up to 3.71% of CD8+T cells reactive with a single tetramer in a minority of subjects. TBC were detected more often in subjects who were infected at the ocular surface, and their presence was associated with infection episodes of longer duration. Detection of C. trachomatis-specific TBC was not associated with the presence of disease or with the estimated load of ocular C. trachomatisinfection at the time of sample collection. High frequencies of C. trachomatis-specific cells did not predict subsequent appearance or resolution of the clinical disease signs of active trachoma.

ABSTRACTThe role of immunity in blinding trachoma is unclear. Conjunctival gene expression was measured in a population where trachoma is endemic. Proinflammatory (tumor necrosis factor alpha and interleukin-1β [IL-1β]), anti-inflammatory (IL-10), and fibrogenic (matrix metalloprotease 9) gene expression was increased in active trachoma. Markers indicative of T-cell response (gamma interferon, IL-4, IL-12p40, and perforin) were increased when chlamydial infection was present.

Recent evidence suggests that eye-seeking flies are important trachoma vectors. We conducted a series of investigations to identify which species of synanthropic flies are potential vector(s) of this blinding disease in The Gambia. Several species of fly were caught in fish-baited attractant traps placed in villages throughout the year (1997/98) but only 2 species, Musca sorbensand M. domestica, were caught from the eyes of children. M. sorbenscomprised <10% of the total number of flies caught with attractant traps but was responsible for >90% of fly-eye contacts, the remainder were made by M. domestica. All fly species were more numerous in the wet season than the dry season. Eyes of young children are considered to be the main reservoir of Chlamydia trachomatis, the causative agent of trachoma. Collections of eye-seeking flies from children showed frequent fly-eye contacts (median [interquartile range], 3 [1·5–7] every 15 min). Children with potentially infective ocular or nasal discharge had twice as many fly-eye contacts than children with no discharge (P< 0·001). There was no difference in exposure to fly-eye contacts if a child sat inside or outside a house (P= 0·273). Female flies were more commonly caught from eyes than male (P< 0·001). The presence of ChlamydiaDNA was demonstrated by PCR on 2 of 395 flies caught from the eyes of children with a current active trachoma infection. Both positive flies were M. sorbens, one male and the other female. Further elucidation of M. sorbensbehavioural ecology and the development of sustainable strategies to control these flies should be a priority. It is likely that M. sorbensis the principal insect vector of trachoma in The Gambia.

Trachoma is initiated during childhood following repeated conjunctival infection with Chlamydia trachomatis, which causes a chronic inflammatory response in some individuals that leads to scarring and in-turning of the eyelids in later life. There is currently no treatment to halt the progression of scarring trachoma due to an incomplete understanding of disease pathogenesis. A cohort study was performed in northern Tanzania in 616 children aged 6 to 10 years at enrollment. Every 3 months for 4 years, children were examined for clinical signs of trachoma, and conjunctival swabs were collected for C. trachomatisdetection and to analyze the expression of 46 immunofibrogenic genes.

To determine if serum antibody response to the 60-kDa chlamydial heat-shock protein (Chsp60) was associated with scarring trachoma, responses to Chlamydia trachomatis and to Chsp60 from 148 Gambian subjects with trachomatous scarring and from 148 controls without clinical evidence of disease from trachoma-endemic communities were characterized. Chsp60 response was found in 32%of cases and 16%of controls (P > .001). Although C. trachomatis titer was also higher in cases than controls, the prevalence of Chsp60 response between the 2 groups remained significantly different after stratifying for C. trachomatis titer (weighted odds ratio [OR] = 2.1, P = .02). Chsp60 response and C. trachomatis serovar A titer of  128 were independently associated with scarring trachoma. The presence of HLA class II allele DRB1*0701 was positively correlated with Chsp60 response (OR = 2.6, P = .02), and DQB1*0301 and DQB1*0501 were negatively associated (OR = 0.42, P < .001; OR = 0.55, P = .46, respectively).

Immune responses to Chlamydia trachomatis contribute to protection from infection and to immunopathologic disease. To test whether subjects' HLA class I (A, B, and Cw) or class II (DRβ1 and DQβ1) types influence risk of trachomatous scarring from chronic infection with C. trachomatis, 153 cases and pair-matched controls in Gambia were studied. No HLA type was associated with protection from scarring, indicating that protective immune responses are not limited to only one or a few HLA-restricted epitopes in C. trachomatis antigens. One class I antigen, HLA-A28, was significantly more common among cases than controls (25.8% vs. 15.9%, respectively; McNemar's odds ratio [OR], 1.88; 95% confidence interval [CI] = 1.01–3.49; P = .046). In DNA subtyping of the A28 specificity, the A *6801 allele was equally common among cases and controls, but the A *6802 allele was significantly overrepresented among cases (McNemar's OR, 3.14; 95% CI = 1.32–7.44; P = .009). This association may be due to an immunopathologic HLA-A *6802-restricted cytotoxic T lymphocyte response.

Direct amplification of the major outer membrane protein (MOMP) gene by polymerase chain reaction (PCR) was used to identify Chlamydia trachomatis in eye swabs from clinically active cases of endemic trachoma in a Gambian village. Chlamydial DNA was detected in 51% of 96 subjects with clinically active disease and in 5% of 37 clinically negative individuals. The PCR detection was combined with typing, using nested primers to variable sequences (VS) 1, 2, and 4 of the MOMP genes to distinguish between trachoma genotypes A, B, and C, respectively. Genotypes A and B were detected in the village, with some individuals harboring both genotypes within the same eye. DNA sequencing revealed strain variants of both genotypes. Typing of genotype and strain variants is now in progress to study trachoma transmission within the village.

Variants of Chlamydia trachomatis in two Gambian villages with hyperendemic trachoma were analyzed by ompl-based polymerase chain reaction and sequencing from conjunctival swabs. Samples collected over a 22-month period included a complete cross-sectional study of each village. Overall, 4 genovar A and 4 B variants were characterized by point mutations in the omp1 gene, resulting in changes in the inferred amino acid sequence. Two genovar A and 2 B variants accounted for 87% of the total ocular chlamydial infection in both villages. Although some flux in the prevalence of individual variants was observed over time, their overall distribution remained remarkably stable. There was no evidence of major antigenic shift arising from recombination events at the amp1 locus as described for genital tract infection. These results indicate that ompl variation in these two trachoma-hyperendemic communities is limited and unlikely to hamper development of trachoma vaccines based on the major outer membrane protein.

In vitro responses of peripheral blood mononuclear cells (PBMC) were measured to antigens of Chlamydia trachomatis in 29 subjects with severe conjunctival scarring and compared with responses from 29 age-, sex-, and location-matched controls from a trachoma-endemic area in The Gambia. Proliferative and interferon-γ responses were measured against chlamydial elementary bodies, recombinant major outer membrane protein, and affinity-purified recombinant chlamydial heat shock protein 60. Serum antibody levels in response to these antigens were also compared between the two groups, and the presence of ocular chlamydial antigen and DNA was assessed. PBMC from donors with conjunctival scarring had reduced proliferative responses to chlamydial antigens but not control antigens or mitogens compared with matched controls. By contrast, subjects with scarring had significantly higher levels of anti-chlamydial IgG antibody compared with matched controls and were more likely to be harboring ocular chlamydial antigen or DNA (or both).