MATERIAL AND METHODS

Bacterial strains and culture

Four C trachomatis serovar E strains isolated from female patients with either a clear symptomatic (S1, S2) or asymptomatic (AS1, AS2) clinical course in both the patient and their partner³ (table 1) were propagated, titrated, and isolated in cyclohexamide treated HeLa cell monolayers using standard techniques.

Murine model, inoculation, and specimen collection

Using a standard model of female genital tract infection,^{4, 5} four groups of 12 progesterone pretreated CF-1 female mice were inoculated intravaginally by direct instillation of 10 μl of bacterial suspension containing approximately 1 × 10⁵ inclusion forming units. Four mice from each strain were euthanised at day 14, the remaining mice at day 56. Eight mice served as non-infected controls. Lower genital tract specimens were obtained by swabbing the vaginal vault and ectocervix with a calcium alginate swab every two to seven days up until day 56. Swabs were placed in transport medium (2-SP) and immediately tested for PMN content before being frozen. Genital tract tissues and local lymph nodes were aseptically isolated, divided into specimens, and stored in 2-SP for culture and polymerase chain reaction (PCR) analysis. All specimens were frozen at −70°C until tested.

Culture, PCR, and PMN responses

The presence of C trachomatis in both the material obtained by swabbing the lower genital tract, and the thawed, homogenised, and centrifugation clarified supernatants from the upper genital tract was determined using previously described culture and PCR techniques.^{3, 5, 6} Finally, lower genital tract inflammation was monitored during the first two weeks of infection by assaying for PMN associated esterase activity (Multistix^® 2 reagent strips; Bayer Corporation, Diagnostic Division, Elkhart, Indiana, USA).

STATISTICAL ANALYSIS

Duration of infection data were analysed using the Wilcoxon rank sum test and the frequency data by χ² or Fisher exact tests where appropriate.

RESULTS

Lower genital tract infection

No significant differences were noted in the incidence or duration of infection either among strains or between the symptomatic and asymptomatic C trachomatis serovar E isolates (table 2). All culture positive samples were also PCR positive, and PCR detected additional positive specimens, both for culture negative time points between two culture positive time points (n  =  14) and after the last culture positive time points (n  =  28). All samples were both culture and PCR negative by day 56. PMN responses in the lower genital tract were also not significantly different either between strains within each group or between the combined data for each group (table 2).

Upper genital tract infection

Progression of the infection into the upper cervix and uterine horns was detected both by culture and, at greater frequency, by PCR, but no differences were noted at day 14 between mice infected with C trachomatis from either symptomatically or asymptomatically infected women, and no signs of gross pathology were visible. All upper genital tract material was negative for C trachomatis on day 56, which was also the case for all lymph nodes and ovaries from both time points. All control mice were negative for C trachomatis by either PCR or culture.

DISCUSSION

Using a well established murine model of C trachomatis female genital tract infection, we showed that there were no differences in the incidence and duration of infection, the lower genital tract PMN response, or the upper genital tract progression, after lower genital tract infection with clinical serovar E isolates from female patients with either a clear symptomatic or asymptomatic course of infection. These experimental data are in agreement with the findings from the most recent human epidemiological study of Geisler et al,¹ suggesting that, at least within a given oculogenital serovar, genetic variation among strains may not strongly contribute to the course of infection. However, it is important to note that our study was not intended specifically to identify possible host or chlamydial factors that might contribute to diverse clinical outcomes. Of interest within the context of our report are the possible roles that newly described polymorphisms within the chlamydial plasticity zone, such as those described in the tryptophan synthase operon,⁷ might play a role in subtle host–pathogen interactions that affect the course of infection, in addition to the stable pattern of serovar prevalence that exists worldwide among clinical oculogenital isolates.

“It seems reasonable to suggest that the focus of future studies to elucidate the basis for differences in clinical course should include analyses of host genetic factors”

Although our study was limited to a single serovar, serovar E is the most prevalent one isolated from genital tract infections, and the lack of variation in the course of infection among these four strains in the murine model suggests that the bacterial background may not have played an important role in the observed clinical differences in the women from whom these strains were isolated. Thus, it seems reasonable to suggest that the focus of future studies to elucidate the basis for differences in clinical course should include analyses of host genetic factors. The study of the host genetic background in relation to disease and infection, called immunogenetics, is a new and rapidly developing field. By determining immune mediators that are important in the susceptibility to infection and the severity of subsequent disease, the assessment of functional mutations in the corresponding genes will potentially lead to the identification of the relevant host factors that contribute to an increased risk for severe disease. For C trachomatis infections important mediators such as interferon γ, interleukin 12 (IL-12), IL-10, and tumour necrosis factor α have been identified using knockout mouse strains and antibody depletion techniques. Based on these and related findings, studies have already been conducted that link an increased risk of pelvic inflammatory disease and tubal infertility with functional polymorphisms in the IL-10 gene and with certain major histocompatibility complex class I alleles and HLA types,^{8, 9} and studies are in progress to determine the possible role of interferon γ, IL-12, and the lipopolysaccharide and heat shock protein responsive Toll-like receptor 4¹⁰ in susceptibility to and severity of C trachomatis genital tract infection.