Population-based analysis of ocular Chlamydia trachomatis in trachoma-endemic West African communities identifies genomic markers of disease severity.

BACKGROUND: Chlamydia trachomatis (Ct) is the most common infectious cause of blindness and bacterial sexually transmitted infection worldwide. Ct strain-specific differences in clinical trachoma suggest that genetic polymorphisms in Ct may contribute to the observed variability in severity of clinical disease. METHODS: Using Ct whole genome sequences obtained directly from conjunctival swabs, we studied Ct genomic diversity and associations between Ct genetic polymorphisms with ocular localization and disease severity in a treatment-naïve trachoma-endemic population in Guinea-Bissau, West Africa. RESULTS: All Ct sequences fall within the T2 ocular clade phylogenetically. This is consistent with the presence of the characteristic deletion in trpA resulting in a truncated non-functional protein and the ocular tyrosine repeat regions present in tarP associated with ocular tissue localization. We have identified 21 Ct non-synonymous single nucleotide polymorphisms (SNPs) associated with ocular localization, including SNPs within pmpD (odds ratio, OR = 4.07, p* = 0.001) and tarP (OR = 0.34, p* = 0.009). Eight synonymous SNPs associated with disease severity were found in yjfH (rlmB) (OR = 0.13, p* = 0.037), CTA0273 (OR = 0.12, p* = 0.027), trmD (OR = 0.12, p* = 0.032), CTA0744 (OR = 0.12, p* = 0.041), glgA (OR = 0.10, p* = 0.026), alaS (OR = 0.10, p* = 0.032), pmpE (OR = 0.08, p* = 0.001) and the intergenic region CTA0744-CTA0745 (OR = 0.13, p* = 0.043). CONCLUSIONS: This study demonstrates the extent of genomic diversity within a naturally circulating population of ocular Ct and is the first to describe novel genomic associations with disease severity. These findings direct investigation of host-pathogen interactions that may be important in ocular Ct pathogenesis and disease transmission.

An index of community ocular Chlamydia trachomatis load for control of trachoma.

Quantitative PCR (Q-PCR) technology has recently been applied to the measurement of ocular loads of Chlamydia trachomatis. We present an index called the community ocular C. trachomatis load (COCTL) which is similar to the community microfilarial load (CMFL) of onchocerciasis. Our index has the advantage of being scale-independent so that, for example, percentage changes are the same whether calculated per eye swab or per Q-PCR capillary. The COCTL for a population or subgroup is formed by adding the arbitrary concentration of 1 organism per ml to each individual Q-PCR quantification, calculating the geometric mean, and finally subtracting 1 per ml again. The use of the COCTL is illustrated in a study of trachoma in northern Tanzania. The COCTL is higher in people with clinical trachoma than those without (5.8 organisms per swab vs. 0.1), and in children aged six months to ten years than in the overall population (1.1 vs. 0.4). The COCTL index is potentially useful for sentinel sites, operational research and calibration of clinical measures of trachoma.

A randomised controlled trial of azithromycin following surgery for trachomatous trichiasis in the Gambia.

BACKGROUND/AIM: Trachomatous trichiasis frequently returns following surgery. Several factors may promote recurrence: preoperative disease severity, surgeon ability, surgical procedure, healing responses, and infection. This study investigates whether enhanced control of infection, both of Chlamydia trachomatis and other bacteria, with azithromycin can improve surgical outcome in a trachoma control programme. METHODS: Individuals with trachomatous trichiasis were examined and operated. After surgery patients were randomised to the azithromycin or control group. The azithromycin group and children in their household were given a dose of azithromycin. Antibiotic treatment was repeated at 6 months. All patients were reassessed at 6 months and 12 months. Samples were collected for C trachomatis polymerase chain reaction and general microbiology at each examination. RESULTS: 451 patients were enrolled. 426 (94%) were reassessed at 1 year, of whom 176 (41.3%) had one or more lashes touching the eye and 84 (19.7%) had five or more lashes. There was no difference in trichiasis recurrence between the azithromycin and control group. Recurrent trichiasis was significantly associated with more severe preoperative trichiasis, bacterial infection, and severe conjunctival inflammation at 12 months. Significant variability in outcome was found between surgeons. Visual acuity and symptoms significantly improved following surgery. CONCLUSION: In this setting, with a low prevalence of active trachoma, azithromycin did not improve the outcome of trichiasis surgery conducted by a trachoma control programme. Audit of trichiasis surgery should be routine.

Long term outcome of trichiasis surgery in the Gambia.

BACKGROUND: Trichiasis surgery is believed to reduce the risk of losing vision from trachoma. There are limited data on the long term outcome of surgery and its effect on vision and corneal opacification. Similarly, the determinants of failure are not well understood. METHODS: A cohort of people in the Gambia who had undergone surgery for trachomatous trichiasis 3-4 years earlier was re-assessed. They were examined clinically and the conjunctiva was sampled for Chlamydia trachomatis polymerase chain reaction (PCR) and general bacterial culture. RESULTS: In total, 141/162 people were re-examined. Recurrent trichiasis was found in 89/214 (41.6%) operated eyes and 52 (24.3%) eyes had five or more lashes touching the globe. Corneal opacification improved in 36 of 78 previously affected eyes. There was a general deterioration in visual acuity between surgery and follow up, which was greater if new corneal opacification developed or trichiasis returned. Recurrent trichiasis was associated with severe conjunctival inflammation and bacterial infection. C trachomatis was detected in only one individual. CONCLUSIONS: Recurrent trichiasis following surgery is a common potentially sight threatening problem. Some improvement in the cornea can occur following surgery and the rate of visual loss tended to be less in those without recurrent trichiasis. The role of conjunctival inflammation and bacterial infection needs to be investigated further. Follow up of patients is advised to identify individuals needing additional surgical treatment.

Host genetic studies in human ocular Chlamydial infection.

Several human and animal models and methods have been used to dissect genetic contributions to immunity and pathogenesis of chlamydial diseases. Considerable achievements have been made in this field of host genetics. The hope is that these studies will lead to medical applications by helping to elicit the function of genes that are involved in host defense against chlamydia and in progression to severe sequelae. In the present article, we review a selection of findings in the forward genetics of ocular Chlamydia trachomatis infection in humans.

Host genetic contribution to the cellular immune response to Chlamydia trachomatis: Heritability estimate from a Gambian twin study.

If the cellular immune response to Chlamydia trachomatis is subject to genetic influences, the degree and mechanisms of such genetic control may have important implications for vaccine development. We estimated the relative contribution of host genetics to the total variation in lymphoproliferative responses to C. trachomatis antigen by analyzing these responses in 64 Gambian twin pairs from trachoma endemic areas. Zygosity was determined by restriction fragment length polymorphism analysis of minisatellite probes and microsatellite typing. Proliferative responses to serovar A elementary body antigen were estimated in monozygotic (MZ) and dizygotic (DZ) twin pairs. We found a stronger correlation and lower within-pair variability in these responses in MZ than in DZ twin pairs. The heritability estimate was 0.39 (P = 0.07) suggesting that host genetic factors contributed 39% of the variation. A better understanding of these genetic influences will contribute to the elucidation of preventive therapies for ocular C. trachomatis infection and may identify important mechanisms in protection for rational vaccine construction.

Genetic variation at the TNF locus and the risk of severe sequelae of ocular Chlamydia trachomatis infection in Gambians.

Tumor necrosis factor (TNF) is thought to be a key mediator of the inflammatory and fibrotic response to Chlamydia trachomatis (Ct) infection. A large matched-pair case-control study investigated putative functional single nucleotide polymorphisms (SNPs) across the major histocompatibility complex (MHC) class III region, including TNF and its immediate neighbors nuclear factor of kappa light polypeptide gene enhancer in B cells (IkappaBL), inhibitor like 1 and lymphotoxin alpha (LTA) in relation to the risk of scarring sequelae of ocular Ct infection. Haplotype and linkage disequilibrium analysis demonstrated two haplotypes, differing at position TNF-308, conferring an increased risk of trichiasis. The TNF-308A allele, and its bearing haplotype, correlated with increased TNF production in lymphocyte cultures stimulated with chlamydial elementary body antigen. Thus TNF-308A may determine directly, or be a marker of a high TNF producer phenotype associated with increased risk of sequelae of chlamydial infection. Multivariate analysis provided evidence for the presence of additional risk-associated variants near the TNF locus.

Temporal cytokine gene expression patterns in subjects with trachoma identify distinct conjunctival responses associated with infection.

Ocular chlamydial disease is clinically diagnosed by the appearance of characteristic inflammatory changes and development of lymphoid follicles in the conjunctiva. Nucleic acid amplification tests and relatively non-invasive methods of sampling the conjunctival surface can be used to quantify the expression of chlamydial and host genes. Using quantitative real-time polymerase chain reaction to detect the presence of Chlamydia trachomatis (CT) 16S rRNA and human interleukin (IL)-1beta, IL-10, IL-12p40, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha transcripts we examined the immune response at the conjunctival surface in a cohort of children living in a trachoma-endemic village in The Gambia. Elevated cytokine transcript levels were associated with the presence of CT 16S rRNA. Subclinical infection (CT infection without clinical signs of disease) elicited an immune response that is proinflammatory in nature, with elevations in the transcription of IL-1beta, IFN-gamma and IL-12p40. Clinically apparent infections were associated with the elevation of mRNA for the multi-functional cytokine TNF-alpha (fibrotic, type 1 inflammatory and regulatory) and the counter regulatory cytokine, IL-10, in addition to the other proinflammatory cytokines. A positive correlation between IFN-gamma transcript levels and the amount of CT 16S rRNA expressed in conjunctiva was found.

Risk of trachomatous scarring and trichiasis in Gambians varies with SNP haplotypes at the interferon-gamma and interleukin-10 loci.

Experimental evidence implicates interferon gamma (IFNgamma) in protection from and resolution of chlamydial infection. Conversely, interleukin 10 (IL10) is associated with susceptibility and persistence of infection and pathology. We studied genetic variation within the IL10 and IFNgamma loci in relation to the risk of developing severe complications of human ocular Chlamydia trachomatis infection. A total of 651 Gambian subjects with scarring trachoma, of whom 307 also had potentially blinding trichiasis and pair-matched controls with normal eyelids, were screened for associations between single-nucleotide polymorphisms (SNPs), SNP haplotypes and the risk of disease. MassEXTEND (Sequenom) and MALDI-TOF mass spectrometry were used for detection and analysis of SNPs and the programs PHASE and SNPHAP used to infer haplotypes from population genetic data. Multivariate conditional logistic regression analysis identified IL10 and IFNgamma SNP haplotypes associated with increased risk of both trachomatous scarring and trichiasis. SNPs in putative IFNgamma and IL10 regulatory regions lay within the disease-associated haplotypes. The IFNgamma +874A allele, previously linked to lower IFNgamma production, lies in the IFNgamma risk haplotype and was more common among cases than controls, but not significantly so. The promoter IL10-1082G allele, previously associated with high IL10 expression, is in both susceptibility and resistance haplotypes.

The sexual health of pupils in years 4 to 6 of primary schools in rural Tanzania.

BACKGROUND/OBJECTIVES: There is an urgent need for effective interventions to improve the sexual and reproductive health of adolescents. Reliable data on the sexual health of adolescents are needed to guide the development of such interventions. The aim was to describe the sexual health of pupils in years 4 to 6 of 121 rural primary schools in north western Tanzania, before the implementation of an innovative sexual health intervention in 58 of the schools. METHODS: A cross sectional survey of primary school pupils in rural Tanzania was carried out. The study population comprised pupils registered in years 4 to 6 of 121 primary schools in 20 rural communities in 1998. Basic demographic information was collected from all pupils seen. Those born before 1 January 1985 (aged approximately 14 years and over) were invited to participate in the survey, and asked about their knowledge and attitudes towards sexual health issues, and their sexual experience. A urine specimen was requested and tested for HIV, Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and, for females, pregnancy. RESULTS: 9283 pupils born before 1 January 1985 were enrolled and provided demographic information and a urine sample. Male pupils were significantly older than females (mean age 15.5 years v 14.8 years, p<0.001), but all other demographic characteristics were similar between the sexes. 14 (0.2%) of the enrolled pupils (four male and 10 female) were HIV positive, 83 (0.9%) were positive for CT, and 12 (0.1%) for NG. 32 female pupils (0.8%) were positive by pregnancy test. Sexual experience was reported by one fifth of primary school girls, and by almost half of boys. Only 45/114 (39%) girls with biological markers of sexual activity reported having had sex. CONCLUSIONS: HIV, CT, NG, and pregnancy were present though at relatively low levels among pupils in years 4 to 6 of primary school. A high proportion of pupils with a biological marker of sexual activity denied ever having had sex. Alternative ways of collecting sensitive data about the sexual behaviour of school pupils should be explored.

Mucosal and systemic immune responses to plasmid protein pgp3 in patients with genital and ocular Chlamydia trachomatis infection.

The circulating and cervical B cell responses to Chlamydia trachomatis plasmid protein pgp3 were characterized in children and adults with ocular or genital chlamydial infection using the enzyme-linked immunospot assay (ELISPOT) and ELISA. No pgp3-specific ASCs were detected in healthy controls, but predominantly IgA ASCs were detected in UK adults with uncomplicated cervicitis or urethritis (P = 0.03, 0.019). In patients with extragenital complications or pelvic inflammatory disease a mixed response with more IgG and IgM ASCs was evident, suggesting a breach of mucosal immune compartmentalization with more extensive infection. In women with chlamydial cervicitis, ASCs secreting predominantly IgA, but also IgG, to pgp3 were present in cervix at presentation, with a frequency 30-50 times higher than blood. Cervical ASC numbers, especially IgG, fell markedly six weeks after antibiotic treatment. We detected principally IgA pgp3-specific antibody secreting cells (ASCs) in children resident in a Gambian endemic area, with a trend towards suppression of IgA responses during intense trachomatous inflammation (P = 0.06), as previously reported for other chlamydial antigens, and in keeping with the findings in genital disease. These data provide a rationale for further studies of immune responses to pgp3 in humans and animal models of chlamydia-induced disease, and its potential use in diagnostic assays and protective immunization strategies.