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Background: The natural history of ocular Chlamydia trachomatis infections in endemic communities has not been well characterised and is an important determinant of the effectiveness of different mass treatment strategies to prevent blindness due to trachoma., Methodology/principal Findings: A multistate hidden Markov model was fitted to data on infection and active disease from 256 untreated villagers in The Gambia who were examined every 2 weeks over a 6-month period. Parameters defining the natural history of trachoma were estimated, and associations between these parameters, demographic and baseline immune measurements examined. The median incubation period following infection was estimated at 17 days (95% confidence interval: 11-28). Disease persisted for longer than infection (median 21 (15-32) weeks) versus 17 (12-24) weeks), with an estimated median duration of post-infection inflammation of 5 (3-8) weeks. The duration of active disease showed a significant decline with age even after accounting for lower rates of re-infection and disease at older ages (p = 0.004). Measurements of levels of baseline IgA to epitopes in the major outer membrane protein of Chlamydia trachomatis were not significantly correlated with protection or more rapid clearance of infection., Conclusions: The average duration of infection with Chlamydia trachomatis especially at younger ages is long. This contributes to the persistence and gradual return of trachoma after community-wide treatment with antibiotics.

Variants of Chlamydia trachomatis in two Gambian villages with hyperendemic trachoma were analyzed by omp1-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 overtime, their overall distribution remained remarkably stable. There was no evidence of major antigenic shift arising from recombination events at the omp1 locus as described for genital tract infection. These results indicate that omp1 variation in these two trachoma-hyperendemic communities is limited and unlikely to hamper development of trachoma vaccines based on the major outer membrane protein.

The application of a diagnostic and genotyping technique based on the polymerase chain reaction (PCR) to the study of trachoma epidemiology in the Gambian village of Jali is reported. PCR based on the major outer membrane protein (MOMP) gene of Chlamydia trachomatis appears to be more sensitive than either isolation or antigen detection by enzyme immunoassay; it had a specificity of 95% and sensitivity of 51% against clinical signs. PCR genotyping identified genotypes A and B of Chlamydia trachomatis circulating in Jali. Sequencing revealed a Pst1 restriction endonuclease site in the amplified MOMP gene of some B strains but not others; Pst1 digestion of the PCR product proved an easy method of distinguishing these strains. The distribution of serotypes and B strain variants shows a significant degree of household clustering (p < 0.001). PCR based genotyping combined with strain typing provides a new and powerful epidemiological tool for the study of transmission events in trachoma.

Polymerase chain reaction (PCR) with primers derived from the sequence of the cryptic chlamydial plasmid was evaluated for the detection of ocular chlamydial infection in trachoma-endemic populations and was used to explore further the relationship between ocular chlamydial infection and clinical signs of trachoma. It was more sensitive than other laboratory techniques, especially for mild cases of trachoma, in detecting infection in 144 (72%) of 200 who fulfilled the World Health Organization case definition. Of 1132 subjects who did not fulfill these criteria, 85 (7.5%) were PCR-positive, but about one-third of these 85 subjects had minor signs of trachoma. Clinically negative subjects who were PCR-positive were more likely than PCR-negative subjects to have acquired signs of disease at 1 and 6 months of follow-up. Clinical signs were twice as likely to have resolved after 1 month in PCR-negative subjects with disease than in those who were PCR-positive.

A single-tube nested PCR able to discriminate between ocular infection with Chlamydia trachomatis of serovar A, B, or C is described. The method uses genotype-specific primers labeled with different fluorochromes, "drop-in/drop-out" PCR amplification, and product analysis on an Applied Biosystems 373A DNA Sequencer using GENESCAN 672 software. The system readily detected mixed infection with serotypes A and B within the same eye. This strategy provides a rapid genotyping method applicable to a wide variety of epidemiological studies.

In order to study the effect of antichlamydial antibodies in ocular secretions on resistance to ocular chlamydial infection and clearance of this infection, we have performed linked longitudinal studies in a Gambian village in which trachoma is endemic. We have measured IgG and IgA antibody levels to a local serotype B isolate of Chlamydia trachomatis by amplified enzyme immunoassay, and chlamydial antigen levels in conjunctival swabs using a commercially available immunoassay which detects chlamydial glycolipid. Having previously demonstrated that sharing a bedroom with a case of active trachoma is a risk factor for acquisition of the disease, we have analyzed the effect of IgG and IgA antibody on the acquisition and persistence of clinical trachoma after controlling for age, sex, exposure to infection and for the presence of chlamydial antigen using a Poisson regression model. We have found that the presence of antichlamydial IgG in ocular secretions of disease-free subjects is associated with an increased incidence of trachoma. IgA antibody shows an opposite trend, but this is not statistically significant. One possible explanation of these findings is that antichlamydial IgG antibodies enhance the infectivity of C. trachomatis for the human eye; this could have major implications for the development of a chlamydial vaccine.

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.

In order to investigate risk factors for the acquisition of trachoma, and to study the effect of continued exposure to ocular chlamydial infection on the severity of inflammatory trachoma and its cicatricial sequelae, a longitudinal study was conducted in a Gambian village. Over a 20-month period, the incidence of active (inflammatory) trachoma was significantly higher among those sharing a bedroom with an active case (64/561, 11.4%) than among those who were not exposed in this way (37/658, 5.6%) (relative risk 1.97, 95% confidence interval 1.33-2.90). There was a positive trend in the odds ratio for severe to moderate inflammatory disease versus mild disease as the number of active cases in the bedroom increased, but this failed to achieve statistical significance (P = 0.0506). Individuals with inflammatory trachoma of moderate or severe intensity at one survey were significantly more likely than others to have moderate or severe inflammatory changes at a previous or subsequent survey (odds ratio 14.9, 95% confidence interval 3.9-68.0), implying that host factors may be more important determinants of severity than the frequency of exposure to reinfection.

Studies on the household distribution of trachoma have reached conflicting conclusions. This paper describes a cross-sectional survey of endemic trachoma in a Gambian village. Cases of active trachoma were mapped, and the compound and household distribution of the disease analysed by a Monte Carlo simulation procedure which takes into account differences in the size and age distribution within individual households. Significant clustering of active trachoma cases both by village compound (p less than 0.0001) and bedroom (less than 0.05) were detected supporting the concept that intra-familial transmission of trachoma is important. There was no evidence of spatial clustering of rooms with higher than expected prevalence of trachoma. Clustering of disease in space or time provides important evidence of infectious aetiology and route of transmission. The methods discussed here are generally applicable in the study of other infectious diseases.

Trachoma control necessitates identification of individuals shedding ocular chlamydiae. Conventional techniques of chlamydial culture are unsuitable for large field surveys in developing countries. In this study an enzyme immunoassay (EIA) developed for the detection of chlamydial antigen in genital-tract infection was used in trachoma. Conjunctival swabs were taken for chlamydial antigen detection from 1225 subjects in a Gambian village with endemic trachoma. Of these, 997 had insignificant or no disease and 172 had mild, 30 moderate, and 26 severe disease. Chlamydial antigen was detected in 5.0%, 20.3%, 46.7%, and 26.9% of subjects in these four categories, respectively. Samples for chlamydial isolation were obtained from 259 subjects. The corresponding rates of detection obtained by chlamydial isolation were 3/90 (3.3%), 16/129 (12.4%), 7/21 (33.3%), and 7/19 (36.8%). If isolation is taken as the "gold standard", the EIA technique had a sensitivity of 70.6% and a specificity of 90%. A substantial proportion of subjects with severe conjunctival scarring, from whom chlamydiae cannot generally be isolated, were antigen positive by EIA, implying that persistent chlamydial antigen plays a part in the pathogenesis of the late sequelae of trachoma.