Prevalence of Chlamydia trachomatis in eye specimens of patients suspected of having viral keratitis: a cross-sectional study

TL;DR

This study found that 8.7% of patients suspected of having viral keratitis actually had Chlamydia trachomatis, with tear fluid being the most effective sample type for detection.

Contribution

The study compares the effectiveness of different eye specimen types for detecting C. trachomatis in keratitis patients.

Findings

C. trachomatis was detected in 8.7% of suspected keratitis patients.

Tear fluid sampling had a 92.9% detection rate, significantly higher than corneal scraping.

Patients under 18 years old had the highest infection rate.

Abstract

Chlamydia trachomatis (C. trachomatis) is a major global health concern, recognized among the leading bacterial causes of sexually transmitted infections and implicated in ocular diseases. Its association with chronic follicular conjunctivitis and severe papillary inflammation underscores the importance of accurate identification in diagnosing trachoma. This study evaluated the prevalence of C. trachomatis in patients suspected to viral keratitis referred to the lab and comparing four different eye specimen types.

This cross-sectional study (2020–2022) involved 161 suspected to viral keratitis patients referred to thet lab and checked for viral and bacterial infections (49.1 % female, 50.9 % male) at Tehran University's Clinical Virology Research Center. Tear fluid, corneal epithelium, and aqueous/vitreous humor samples were analyzed using the Qiagen Mini Blood Kit for DNA extraction and Multiplex Real-Time PCR with the Fast-track diagnostics/SIEMENS eye kit. C. trachomatis was detected in 14 patients (8.7 %), who showed clinical features such as follicular conjunctivitis, corneal neovascularization, epithelial erosions, and conjunctival scarring. Details on pathology, disease course, treatments, and outcomes are provided in.

This study highlights the prevalence of C. trachomatis in suspected keratitis cases, offering a comparative view across different eye specimen types. Accurate detection using molecular assays supports timely intervention and targeted treatment, improving diagnostic precision and patient outcomes.

•C. trachomatis prevalence among suspected keratitis patients was 8.7 %.•Infection rates were 11.4 % in females and 6 % in males.•Tear fluid sampling outperformed corneal scraping in detection (92.9 % vs. 21.4 %).•Among different age groups, those under 18 exhibited the highest infection rate.

C. trachomatis prevalence among suspected keratitis patients was 8.7 %.

Infection rates were 11.4 % in females and 6 % in males.

Tear fluid sampling outperformed corneal scraping in detection (92.9 % vs. 21.4 %).

Among different age groups, those under 18 exhibited the highest infection rate.

Full text

Introduction

1

Chlamydia trachomatis (C. trachomatis) is a major sexually transmitted pathogen and one of the most prevalent bacterial infections globally. Of its 15 serovars, four (A, B, Ba, and C) can cause ocular infections, including trachoma and inclusion conjunctivitis, underscoring their significant impact on public health [1].

Trachoma, a leading cause of infectious blindness worldwide, is caused by C. trachomatis and marked by follicle formation from lymphocyte infiltration beneath the eye surface. It remains endemic in resource-limited regions of Africa, Asia, and South America, especially in countries like India, Ethiopia, and Nigeria. [2,3]. In Iran, trachoma is relatively rare, and most keratitis cases are not attributed to C. trachomatis. Following the World Health Organization (WHO) through the year 2022, 125 million people were settled in trachoma endemic areas and were threatened by blindness [4].

C. trachomatis is a known cause of ocular infections, with repeated conjunctival infections leading to chronic follicular conjunctivitis (TF) on the upper eyelid. Severe cases show intense papillary inflammation (TI), and active trachoma is defined by the presence of both TF and TI [5].

Advancements in molecular biology, particularly molecular assays, have transformed the diagnosis of many infectious agents across various clinical samples. In trachoma, accurate pathogen identification is essential to prevent complications and ensure effective treatment.

The objective of the study was to evaluate the prevalence of C. trachomatis in patients suspected to keratitis with comparative view of four different eye specimen types.

Material and Methods

2

Study population

2.1

This prospective cross-sectional study (2020–2022) was conducted at Tehran University's Clinical Virology Research Center. Based on an estimated C. trachomatis prevalence of 10 % in suspected keratitis cases, a sample size of 138 was calculated (95 % confidence, 5 % margin of error). To ensure adequate power, 161 patients (79 females, 82 males) were recruited and examined by ophthalmologists.

Suspected keratitis cases were identified based on symptoms like eye pain, photophobia, vision loss, redness, and tearing, along with clinical signs such as corneal opacity, epithelial defects, or stromal infiltration. Ophthalmologists also reviewed histories of ocular trauma, contact lens use, prior eye infections, or related systemic infections to confirm eligibility and proceed with specimen collection.

Sample collection and genome extraction

2.2

Samples were collected from the affected eye, including tear fluid, corneal epithelium, aqueous humor, and vitreous humor, under sterile conditions. A total of 87 right eyes and 74 left eyes were sampled. Ophthalmologists performed deep corneal scrapings using sterile stainless-steel blades under a slit lamp. Tear fluid was obtained by rinsing the ocular surface with 500 μl of saline three times. For epithelial keratitis, corneal epithelium was scraped from the ulcer's edge with plastic swabs. Aqueous humor (0.1 μl) was collected via anterior chamber paracentesis with a 27-gauge needle for stromal or endothelial keratitis. Vitreous humor was also sampled. The specimens, stored in 2 mL of Phosphate Buffer Saline (PBS), were transported to the lab.

Using the Qiagen Mini Blood Kit (Qiagen, Hilden, Germany) and following the manufacturer's guidelines, bacterial nucleic acids were extracted from 100 μl of each specimen. Additionally, the concentration of the extracted genome was evaluated using nanodrop at A 260/280, aiming for an A ratio of approximately 1.8.

Real-time PCR

2.3

Real-time PCR was performed using the Fast-track diagnostics/SIEMENS eye kit (Esch-sur-Alzette, Luxembourg) to detect C. trachomatis. The multiplex PCR targets the 16S rRNA gene, which is conserved across all Chlamydia species for reliable detection. The assay amplifies a 120 base pair segment using the following primers: Forward 5′-AGCAGGAGGAGTGAAGGTG-3′ and Reverse 5′-GCCAGCAGAGTGGTGTTAC-3’.

PCR reactions were performed in a 5 μL volume with thermal cycling: initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and extention at 72 °C for 30 s. Quantification of the PCR product was assessed by measuring the quantification cycle (Cq) value. A Cq value < 35 was considered positive, while >35 was negative.

The primer set targeting the 16S rRNA gene is specific to C. trachomatis, minimizing cross-reactivity and improving diagnostic accuracy. Validated by multiple studies and manufacturer data, the assay has a sensitivity of 96–100 % and specificity of 98–100 %, making it reliable for detecting C. trachomatis in ocular samples.

Statistical analysis

2.4

Data were analyzed using STATA (ver. 17) to assess Chlamydia infection, comparing positive test rates across sex and age groups with independent t-tests. Odds Ratio (OR) was calculated to compare group effects, with a significance level of 0.05. After statistical description, the reliability and accuracy of the findings were evaluated.

Results

3

Demographic data

3.1

The study included 161 patients (82 men, 79 women), with 33 (20.5 %) under 18, 50 (31.1 %) aged 18–49, and 78 (48.4 %) over 50. Of these, 14 (8.7 %) tested positive for C. trachomatis, and 147 (91.3 %) tested negative. The highest detection rate was in tear fluid (92.9 %). In terms of eye laterality, 8 infections were from the right eye and 6 from the left. No reinfections were noted. Alternative diagnoses for negative cases included Staphylococcus aureus (38), Pseudomonas aeruginosa (22), fungal keratitis (17), and other bacterial or idiopathic keratitis (70).

Prevalence of C. trachomatis

3.2

We analyzed the prevalence of C. trachomatis infection across the entire cohort, as well as separately for men and women. Subsequently, we conducted detailed statistical analyses, as summarized in Table 1, Table 2.Table 1. The proportions and the test of the association between infection and gender.Table 1variablePositiveNegativesignificantSexMale5 (6 %)77 (94 %)0.2130.116Female9 (11.4 %)70 (88.6 %)Table 2. The proportions and the test of the association between infection and age categories.Table 2variablePositiveNegativeSignificant<185 (15 %)28 (85 %)0.038∗Age18–492 (4 %)48 (96 %)0.424≥507 (9 %)71 (91 %)0.140

In the male subgroup, 5 out of 82 (6 %) tested positive, while 9 out of 79 women (11.4 %) tested positive. The infection rate was 5.4 % higher in women, but this difference was not statistically significant (P_value_ = 0.116). The Odds Ratio (OR) of infection between women and men was 1.98, indicating women are nearly twice as likely to be infected, though this difference was not statistically significant (P_value_ = 0.24).

In the age group under 18, 5 out of 33 (15 %) tested positive, while 28 (85 %) tested negative. The group aged 18–49 had the lowest infection rate, serving as the baseline. Compared to this group, the under-18 group showed a 0.11 higher infection rate, statistically significant (P_value_ = 0.038). The over-50 group had a 0.05 higher rate, but this difference was not significant (P_value_ = 0.140). Detailed results are in Table 2, Table 3.Table 3. Sampling methods and number of diagnosis.Table 3. SampleFrequency of diagnosisPercent of diagnosisTear fluid1392.9 %Corneal epithelium321.4 %Aqueos Humor00 %Vitreous Humor00 %

Detection in different samples

3.3

We assessed the effectiveness of three sampling techniques for detection, with results in Table 3. Corneal epithelium identified 21.4 % of positive cases, while tear fluid detected 92.9 %. The 71.5 % difference in detection efficacy was statistically significant (P_value_ = 0.0001).See Table 4

Risk factors among patients

3.4

Among the 161 patients, 47 (29.2 %) used contact lenses, 18 (11.2 %) had recent ocular trauma, and 23 (14.3 %) had used topical corticosteroids in the past month. Other risk factors included prior ocular infection (19 patients, 11.8 %) and systemic diseases with ocular involvement (11 patients, 6.8 %). These factors were present in both C. trachomatis-positive and -negative patients, with no statistically significant associations found.

Discussion

4

The analysis showed tear fluid samples had the highest positive infection rate, followed by corneal epithelium. This suggests tear fluid is the most reliable for detecting C. trachomatis in corneal infections, and clinicians should prioritize these samples for accurate diagnosis and treatment. Tear fluid samples yielded the highest detection rate for C. trachomatis, with no reinfections identified. PCR showed consistent sensitivity across all sample types. As detailed in Table 4, patients with C. trachomatis keratitis exhibited diverse clinical features, including follicular reactions, chronic conjunctivitis, and pannus formation, with some having bilateral involvement or a relapsing course, indicating the chronic nature of chlamydial ocular infections. These findings align with previous data [6], highlighting the importance of PCR testing to differentiate C. trachomatis from other microbial causes, especially in atypical or treatment-resistant cases.Table 4. Clinical characteristics of patients diagnosed with Chlamydia trachomatis keratitis (n = 14).Table 4. Patient IDAgeSexLateralityPathology FindingsProgressionTreatment AdministeredTreatment ResponseP0125FUnilateralFollicular conjunctivitis, stromal infiltrationSubacute, gradual worseningTopical azithromycin + oral doxycyclineComplete resolutionP0232MBilateralPannus formation, epithelial defectChronic, relapsingOral doxycycline + lubricantsPartial improvementP0318FUnilateralSuperficial punctate keratitisAcute onset, stableTopical erythromycinFull recoveryP0445MBilateralSubepithelial hazeChronicOral azithromycin + artificial tearsMild improvementP0529FUnilateralMild stromal edemaSubacuteTopical azithromycinFull recoveryP0637MUnilateralPeripheral corneal neovascularizationChronic, progressiveOral doxycycline + topical steroidsPartial improvementP0722FBilateralConjunctival scarringChronicOral azithromycinStableP0831MUnilateralFollicular hypertrophyAcuteTopical tetracyclineFull recoveryP0927FBilateralMixed infiltratesRelapsingOral doxycycline + topical erythromycinSignificant improvementP1040MUnilateralScattered epithelial erosionsAcute onsetTopical azithromycinFull recoveryP1133FBilateralChronic conjunctivitisProgressiveOral doxycyclineModerate improvementP1226MUnilateralSuperficial haze, no ulcerationSubacuteTopical erythromycinFull resolutionP1319FUnilateralFollicular conjunctivitisAcuteTopical tetracyclineFull recoveryP1436MBilateralConjunctival inclusion bodiesChronicOral azithromycin + corticosteroidsMild improvement

Although C. trachomatis is traditionally associated with ocular infections, its role in keratitis may be limited. Only 8.7 % of our cohort tested positive, a figure consistent with literature suggesting C. trachomatis is not the leading cause of keratitis [7]Our findings reinforce that in Iran, common etiologies include Staphylococcus aureus, Pseudomonas aeruginosa, and fungi. Results from 161 subjects of varying ages indicated a higher prevalence in individuals over 50 and under 18, with noticeably higher infection rates in females.

A review by Teweldemedhin et al. on bacterial ocular infections highlights key pathogens linked to various diagnoses, including Staphylococcus aureus, Coagulase-negative Staphylococci, Chlamydia trachomatis, Streptococcus pneumoniae, and Pseudomonas aeruginosa. The causative bacteria vary by condition such as blepharitis, conjunctivitis, dacryocystitis, keratitis, and endophthalmitis underscoring the need for accurate pathogen identification and preventive strategies [8].

Sharma et al. studied C. trachomatis eye infections by analyzing conjunctival swabs from 1281 patients. Antigen positivity ranged from 22 to 28 %, with higher rates in children under 11 and adults over 60. Females aged 31–60 had significantly higher positivity than males. The findings highlight a persistent presence of C. trachomatis in Northern India, challenging earlier perceptions. [9]. A study in Kongwa, Tanzania, examined trachoma prevalence in children as part of a control initiative. PCR-EIA detected Chlamydia in 49 % of conjunctival scrapes, compared to 22 % by DFA. Following topical tetracycline treatment, chlamydial DNA levels dropped, but some cases persisted, indicating possible antibiotic resistance [10]. Macleod et al. conducted a population-based survey of 2306 subjects in Fiji's Western Division to assess ocular C. trachomatis prevalence. Clinical trachoma was found in 3.4 % of cases, while 1.9 % of children aged 1–9 tested positive for ocular C. trachomatis infection. [11]. Also, Nash et al. assessed ocular C. trachomatis prevalence in Ethiopia's Amhara Region, reporting an overall rate of 4.7 %, with variation across the 10 administrative zones [12]. Compared to our study, we showed higher infection rate. Our study included older patients which showed high infection rate and that may be a reason for its elevated prevalence compared to this study.

Although the study had a statistically adequate population, further research with a larger sample is recommended. Limitations include the relatively small sample size, absence of comprehensive microbial testing in cases, and lack of long-term follow-up to assess reinfection or treatment outcomes.

Conclusion

5

The study found a low prevalence of C. trachomatis in keratitis-suspected patients, suggesting a re-evaluation of its role in keratitis and ocular infections, particularly in non-endemic regions like Iran. The findings highlight varying infection rates across age groups, with a significant difference in those under 18. While gender and older age group differences were not statistically significant, tear fluid showed heightened sensitivity in detecting C. trachomatis. Future research should expand diagnostic panels and focus on age-specific interventions for better management of C. trachomatis infections.

CRediT authorship contribution statement

Arash Letafati: Supervision. Parsa Ghafari: Methodology. Niloofar Mobarezpour: Writing – original draft, Methodology. Mohammad Haddadi: Methodology, Investigation. Mersedeh Arbabinia: Methodology. Zahra Rostami: Methodology. Yasamin Meamarzadegan: Writing – review & editing, Writing – original draft, Investigation, Data curation. Aniseh Dadgar: Formal analysis. Zahra Tayebi: Writing – review & editing, Validation, Supervision.

Ethics approval and consent to participate

The study was performed following the Declaration of Helsinki and the present study protocol was approved by the Ethics Committee of Tehran University of Medical Sciences, Tehran, Iran.

Funding/support

This study is supported by 10.13039/501100004484Tehran University of Medical Sciences.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.