A suitable and efficient optimization system for the culture of Chlamydia trachomatis in adult inclusion conjunctivitis

Abstract

The prevalence of Chlamydia trachomatis infection in the genitourinary tract is increasing, with an annual rise of 9 million cases. Individuals afflicted with these infections are at a heightened risk of developing adult inclusive conjunctivitis (AIC), which is commonly recognized as the ocular manifestation of this sexually transmitted infection. Despite its significant clinical implications, the lack of distinctive symptoms and the overlap with other ocular conditions often lead to underdiagnosis or misdiagnosis of AIC associated with C. trachomatis infection. Here, we established six distinct C. trachomatis culture cell lines, specifically highlighting the MA104 N*V cell line that exhibited diminished expression of interferon regulatory factor 3 (IRF3) and signal transducer and activator of transcription 1 (STAT1), resulting in reduced interferons. Infected MA104 N*V cells displayed the highest count of intracytoplasmic inclusions detected through immunofluorescence staining, peaking at 48 h postinfection. Subsequently, MA104 N*V cells were employed for clinical screening in adult patients diagnosed with AIC. Among the evaluated cohort of 20 patients, quantitative PCR (qPCR) testing revealed positive results in seven individuals, indicating the presence of C. trachomatis infection. Furthermore, the MA104 N*V cell cultures derived from these infected patients demonstrated successful cultivation and replication of the pathogen, confirming its viability and infectivity. Molecular genotyping identified four distinct urogenital serovars, with serovar D being the most prevalent (4/7), followed by E (1/7), F (1/7), and Ia (1/7). This novel cellular model contributes to studies on C. trachomatis pathogenesis, molecular mechanisms, and host-pathogen interactions both in vitro and in vivo. It also aids in acquiring clinically relevant strains critical for advancing diagnostics, treatments, and vaccines against C. trachomatis.

Keywords: Chlamydia trachomatis; adult inclusion conjunctivitis; ompA genotyping; sexually transmitted disease.

Figure 1.

Schematic diagram of conjunctival sac swab sampling, transport, storage, and culture in clinical patients.

Figure 2.

Growth and characterization of six epithelial cell lines for culture of C. trachomatis. (A) Morphology of MA104, MA104 N*V, HCEC, Hela, BGMK, and Vero cells under contrast phase microscope. Scale bar = 100 µm. (B) Immunofluorence staining of STAT1 and IRF3 within MA104 and MA104 N*V cells. Nuclei were counterstained with DAPI. Scale bar = 20 µm. (C) Western blotting demonstrating the decreased expression of STAT1 and IRF3 in MA104 N*V cells. GAPDH was used as a loading control. (D) RT-qPCR showing the significantly decreased expression levels of STAT1 and IRF3 in the MA104 N*V cell line (**P < .01, ***P < .001).

Figure 3.

Assessments of infectivity of C. trachomatis across six epithelial cell lines. (A) Observation of six distinct cell lines 48 h post C. trachomatis infection using IF staining. Scale bar = 20 µm. (B) Comparison of the number of intracytoplasmic inclusions in six different cell lines infected with 1000 and 5000 IFU of C. trachomatis. The number of intracytoplasmic inclusions were assessed in 20 randomly selected fields at 48 h postinfection (***P < .001). (C) Number of intracytoplasmic inclusions in cells infected with 5000 IFU of C. trachomatis. At 24, 48, and 72 h postinfection, 20 randomly selected fields were assessed for the number of intracytoplasmic inclusions in IF staining (comparison with HeLa cells, *P < .05, **P < .01, ***P < .001, and ns: nonsignificant). (D) C. trachomatis cultures were harvested and quantified using qPCR at 24, 48, and 72 h. Statistical comparison with HeLa cells revealed significant differences (*P < .05). (E) Scatterplot showing the strong positive correlation between the number of intracytoplasmic inclusions and the C. trachomatis DNA copy number. Pearson correlation was used.

Figure 4.

Slit-lamp images from seven confirmed AIC cases, accompanied by laboratory test in MA104 N*V cells with IF and Giemsa staining. The arrow indicates typical C. trachomatis intracytoplasmic inclusions detected under the microscope. Scale bar = 50 µm.

Figure 5.

Maximum-likelihood phylogenetic tree displaying the evolutionary relationships of C. trachomatis ompA gene sequences. The leaves of the tree are color-coded according to genotype. GenBank accession numbers of all available reference sequences, indicated in parentheses, uniquely identify each sequence in the analysis.