Table of Contents  

Abd El-Aal, El Saied, El Sayed, Foad, Abd Elazeim, Mashaly, and Ahmed: Ocular Chlamydia trachomatis in a tertiary hospital

Introduction

Chlamydia trachomatis is an obligate intracellular bacterium and is known to infect human tissue by direct spread from other infected humans or fomites. In Chlamydia infection, the elementary body is the infectious form and it is very small and can easily escape host defences and invade epithelial cells where it begins to multiply. Chlamydia is known to cause both humoral and cellular immune responses in trachoma.1

The surface of Chlamydia is composed of a complex molecular mosaic of lipopolysaccharide (LPS) and outer membrane proteins. The most dominant component is the major outer membrane protein, which represents 60% of the dry weight of the outer membrane.2

Chlamydia trachomatis is the most common cause of chronic follicular conjunctivitis. The organism causes three clinical syndromes: trachoma, adult inclusion conjunctivitis and neonatal conjunctivitis.3

Trachoma is the most common infectious cause of blindness, which results from serotypes A–C. It is endemic in many parts of the world and is especially prevalent in areas where there is close human contact and poor hygiene. Active trachoma is characterized by the presence of subepithelial follicles (trachomatous follicular) and/or (trachomatous intense). After years of repeated reinfection, scarring may occur, which can lead to distortion of the eyelid, causing the eyelashes to turn inwards (trichiasis) and scratch the cornea, resulting in corneal opacity and blindness.4

Adult inclusion conjunctivitis results from C. trachomatis serotypes D–K, which cause a chronic follicular conjunctivitis that can occur in adults or in the neonate. The disease in adults is transmitted venereally or via hand-to-eye contact.5

Various laboratory assays can be used for the diagnosis of ocular C. trachomatis infection, including examination of stained conjunctival scrapings for intracytoplasmic inclusions, tissue culture, immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and nucleic acid amplification, such as polymerase chain reaction.4

Surgery for inturned lashes, antibiotics for active disease, facial cleanliness and environmental improvement (SAFE) strategy is used for the control of trachoma. A single oral dose of the antibiotic azithromycin is as effective as 6 weeks of topical tetracycline in trachoma control. Azithromycin has unique pharmacokinetic properties that make it ideal for treating trachoma: good oral bioavailability and distribution to tissues, sustained high tissue levels with low protein binding and high intracellular concentration, which is important in treating C. trachomatis, and the safety of three dose regimen (once per week for three weeks) or single dose has been demonstrated in clinical trials.6

By means of the SAFE strategy, the World Health Organization (WHO) and its partners aim to eliminate trachoma as a public health problem by the year 2020.4 Thus, our aim was to assess active trachoma and actual ocular C. trachomatis infection by its isolation in the Vero cell line, then its detection by Giemsa and iodine stains, ELISA and Gen-Probe.

Materials and methods

This study included 40 patients clinically diagnosed with chlamydial conjunctivitis, collected from the outpatient clinic of the Ophthalmology Centre, Mansoura University Hospital (a tertiary hospital), Egypt. They were suspected of having follicular conjunctivitis in the form of redness, conjunctival hyperaemia and subepithelial follicles. An informed written consent was obtained from all participants, and the Ethics Committee of Mansoura University Hospital approved the study.

Sample processing

Using standard techniques under topical anaesthesia, two scrapings were collected from the affected conjunctiva under slit lamp magnification with sterile blade (no 15) on a Bard–Parker handle. One scrape was fixed on a glass slide for Giemsa stain (Sigma-Aldrich, St Louis, MO, USA) and the other scrape was transported to the National Research Centre (Cairo, Egypt) in sucrose phosphate transport medium [8 mM KH2PO4, 12 mM K2HPO4, 0.2 M sucrose (all from Merck KGaA, Darmstadt, Germany), 2.5 µg/ml amphotericin B (Fungizone®, Flow Laboratories Ltd., Irvine, UK), 50 µg/ml streptomycin and 100 µg/ml vancomycin].7

Cell culture

The Vero cell line was cultured to form a monolayer in a tissue culture flask. The procedure was carried out under a vertical laminar air filter hood. The growth medium was then removed and the cell monolayer washed twice with sterile phosphate buffered saline (pH 7.5) (Sigma-Aldrich) prewarmed to 37°C. The cells were dissociated by adding 5–6 ml of sterile 0.25% trypsin EDTA (ethylenediaminetetraacetic acid) (Sigma-Aldrich) to cover the cell sheet and incubated for 1–2 minutes at 37°C, followed by removal of the excess trypsin. The flask was examined under the inverted microscope from time to time to observe cell dissociation. The cells were then resuspended in 10 ml Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS) (Thermo Fisher Scientific, Renfrew, UK)(pH 7.4, adjusted with NaHCO3), amphotericin B 2.5 µg/ml and gentamicin 10 µg/ml. The resuspended cells were distributed on tissue culture plates and incubated at 37°C in a CO2 incubator until a complete monolayer sheet formed (within 48 hours). The growth medium was replaced by maintenance medium (2% FCS) and the plates used for inoculation with the test samples. Tissue culture plates (Caster 24-well plates; Sigma-Aldrich) were prepared by seeding each well with 0.5 ml of the cell suspension. A 200-µm aliquot of the specimen was inoculated into the tissue culture plate well and two non-inoculated tissue culture wells were used as negative controls for each set of specimens. The infected tissue culture plates were incubated at 37°C in a CO2 incubator. The maintenance medium of each well was changed 24 hours after inoculation and after 1 week. The infected tissue culture plates were examined daily by inverted microscope for the specific cytopathic effect and the samples showing cytological changes were frozen and thawed three successive times to extract the expected pathogen in the infected cells. We collected the supernatant and used Giemsa and iodine stains, Gen-Probe and ELISA to identify the bacterium.

Giemsa stain

The air-dried smear or the cell monolayer were fixed in absolute methanol for 5–10 minutes and dried, and smears were stained for 20 minutes using Giemsa solution (1 ml + 19 ml distilled water), then rinsed in water buffered at pH 7.2. Slides were passed quickly through 95% ethyl alcohol or methanol, rinsed in buffer, then dried and examined by light microscope. C. trachomatis inclusions were early acidophilic intracytoplasmic, late were basophilic.

Iodine stain

The cell monolayer was fixed in absolute methanol or in 10% (v/v) formalin (4% formaldehyde) saline and stained in 5% iodine in 10% potassium iodide (all products from Sigma-Aldrich) for 5 minutes.8 A cover slip was placed over the slide, which was examined as a wet mount. Inclusions appear as dense collections of small, round deeply stained particles within a defined area in the cytoplasm, deep brown/black when stained by iodine.

Enzyme-linked immunosorbent assay for Chlamydia (MASTAZYM-Chlamydia, United Kingdom)

The test utilizes a mouse monolayer IgG antibody specific to C. trachomatis LPS for antigen capture.

Gen-Probe

The Gen-Probe PACE 2 assay (Gen-Probe, San Diego, California, USA) uses a single-stranded DNA probe with a chemiluminescent label that is complementary to the ribosomal RNA of the target organism. After the ribosomal RNA is released from the organism, the labelled DNA probe combines with the target organism’s ribosomal RNA to form a stable DNA–RNA hybrid. The labelled DNA–RNA hybrid is separated from the non-hybridized probe and is measured in a Gen-Probe Leader luminometer (Gen-Probe). The test result is calculated as the difference between the response of the specimen and the response of the negative reference.

Statistical analysis

Data were analysed using Statistical Package for Social Sciences (SPSS), version 10. Qualitative data were presented as number and percentage. Quantitative data were presented as mean and standard deviation (SD). The chi-squared test (χ2) was used to find the association between variables of qualitative data. A P-value of < 0.05 indicates a significant result. Validity tests were carried out using sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and efficiency.

Results

This study included 40 conjunctivitis patients: 23 males and 17 females. The mean age was 32.3 ± 15.9 (SD) years. Most of them were from rural areas, 34/40 (85%) (Table 1). There was recurrence in two of the four positive samples (50%). The mean duration of illness was 3.1 ± 2.7 (SD) days with a range of 1–15 days.

TABLE 1

Demographic data of the studied patients (n = 40)

Data Mean ± SD (range) or number
Age (years) 32.3 ± 15.9 (21–59)
Gender
Males 23, 57.5%
Females 17, 42.5%
Residence
Rural 34, 85%
Urban 6, 15%

Chlamydia trachomatis was detected by direct Giemsa stain in 1/40 (2.5%) of samples but isolated by culture using the Vero cell line in 4/40 samples (10%) (Table 2). C. trachomatis was identified in culture by Giemsa and iodine stains and Gen-Probe (100%). However, ELISA identified only 50% of positive samples (Table 3 and Figure 1).

TABLE 2

Detection of C. trachomatis infection by direct Giemsa stain and culture in the studied patients (n = 40)

Diagnostic method Conjunctivitis (n = 40)
Number %
Direct Giemsa stain (PMNs + inclusion bodies)
Positive 1 2.5
Negative 39 97.5
Culture
Positive 4 10
Negative 36 90

PMN, polymorphonuclear leucocyte.

TABLE 3

Evaluation of diagnostic tests for C. trachomatis after culture

Diagnostic method Result Culture identification
Number % from positive samples (n = 4) % from total samples (n = 40)
Giemsa Positive 4 100 10.0
Iodine Positive 4 100 10.0
ELISA Positive 2 50 5.0
Gen-Probe Positive 4 100 10.0
FIGURE 1

C. trachomatis identification by iodine (A, B) and Giemsa stain (C, D) after culture.

8-2-4-fig1a.png8-2-4-fig1b.png8-2-4-fig1c.png8-2-4-fig1d.png

Direct Giemsa stain provides low sensitivity (25%) but a high specificity (100%) compared with cell culturing. Results for PPV, NPV and efficiency were 100%, 92.3% and 92.5%, respectively, with high statistically significant differences between both tests (P-value < 0.001) (Table 4).

TABLE 4

Validity of direct Giemsa stain for diagnosis of C. trachomatis in comparison with cell culture (n = 40)

Direct Giemsa C. trachomatis culture
Positive Negative Total Sensitivity Specificity PPV NPV Efficiency P-value
No % No % No % % % % % %
Positive 1 25 0 0 1 2.5 25 100 100 92.3 92.5 < 0.001
Negative 3 75 36 100 39 97.5
Total 4 10 36 90 40 100

Table 5 shows the validity of C. trachomatis culture identification by Gen-Probe in relation to identification via Giemsa stain. Of the samples, 4/40 (10%) were positive for C. trachomatis using the two methods, so all the indices for Gen-Probe as compared with Giemsa were 100% with high statistically significant differences between both tests (P-value < 0.001).

TABLE 5

Validity of C. trachomatis culture identification by Gen-probe in comparison with Giemsa stain identification (n = 40)

C. trachomatis identified by Gen-Probe C. trachomatis identification by Giemsa
Positive Negative Total Sensitivity Specificity PPV NPV Efficiency P-value
No % No % No % % % % % %
Positive 4 100 0 0 4 10 100 100 100 100 100 < 0.001
Negative 0 0 36 100 36 90
Total 4 10 36 90 40 100

Table 6 shows the validity of C. trachomatis culture identification by ELISA in comparison with identification via Giemsa stain. From the 4/40 (10%) positive culture samples identified by Giemsa, only 2/40 (5%) were positively identified by ELISA. Sensitivity, specificity, PPV, NPV and efficiency of ELISA, as related to Giemsa, were 50%, 100%, 100%, 94.7% and 95%, respectively, with high statistically significant differences between both tests (P-value < 0.001).

TABLE 6

Validity of C. trachomatis culture identification by ELISA in comparison with Giemsa stain identification (n = 40)

C. trachomatis identified by ELISA C. trachomatis identified by Giemsa
Positive Negative Total Sensitivity Specificity PPV NPV Efficiency P-value
No % No % No % % % % % %
Positive 2 50 0 0 2 5 50 100 100 94.7 95.0 < 0.001
Negative 2 50 36 100 38 95
Total 4 10 36 90 40 100

Discussion

Trachoma remains the leading infectious cause of blindness worldwide. WHO recommends mass antibiotic distributions in its strategy to eliminate blinding trachoma.9

Chlamydial recurrence was 50% in the studied patients, which agrees with Dawson et al.,10 who reported that there was constant reinfection with C. trachomatis.

Tissue culture sensitivity is less than 100%, expensive and time-consuming, but remains an asset for the laboratory diagnosis of chlamydial infection because of its near-perfect specificity. It proves that viable organisms (rather than just nucleic acid) are present in the sample and, in specialized laboratories, allows the antimicrobial sensitivities of C. trachomatis isolates to be elucidated. Although resistance in C. trachomatis has not yet proven to be a clinical problem, there is potential for it to develop with extensive use of antibiotics. Because annual mass distribution of single-dose azithromycin is now a strategy for trachoma control in some countries, sensitivity testing may be of increasing relevance to policy-makers.4

The Vero cell line was used for the isolation of C. trachomatis, as performed previously by Rogers and Andersen11 and Vanrompay et al.12 for the propagation of C. trachomatis.

In our study, direct Giemsa stain (PMNs and intracytoplasmic inclusions bodies) was positive in 1/4 (25%). Madhavan et al.13 reported a slightly greater percentage (36%), but here we isolated C. trachomatis by culture in 4/40 samples (10%). C. trachomatis was identified in culture by Giemsa and iodine stains and Gen-Probe (100%). However, ELISA identified only 2/4 (50%) of positive C. trachomatis samples. Tabrizi et al.14 reported similar prevalence by culture (13.9%), others, Talley et al.15 and Jespersen et al.,16 reported lower prevalences (6.7% and 6%, respectively). However, Stenberg et al.17 reported greater C. trachomatis prevalence in culture (39%). This controversy in the results may be because of population differences. The microplates used in our techniques are considered more economical than the shell vials and are equivalent in sensitivity.18

In our study, sensitivity of direct Giemsa stain in relation to tissue culture was 25%. Kobayashi et al.19 and Madhavan et al.13 reported that traditional cytological investigation is insensitive and subjective.

We compared the identification of C. trachomatis by Gen-Probe or ELISA with Giemsa stain because Giemsa and iodine stains or immunofluorescence are considered gold-standard techniques for identification of Chlamydia.20

All the validity indices for Gen-Probe identification of C. trachomatis culture as related to Giemsa stain identification were 100%, as shown in Table 5. Similarly, Warren et al.,21 Miettinen et al.22 and Moncada et al.23 reported Gen-Probe sensitivities of 95.8%, 90% and 99.4%, respectively.

In this study, the sensitivity of ELISA for the identification of C. trachomatis was low (50%) compared with Giemsa stain. Similarly, Wylie et al.24 and Tantisira et al.25 recorded sensitivities of 63.4% and 40%, respectively. This lower sensitivity of the ELISA may be because of the high detection limit of the kit.

From this study we concluded that C. trachomatis was still detected at a relatively high rate in acute follicular conjunctivitis patients as detected with culture on the Vero cell line. Identification of C. trachomatis in culture was 100% by Giemsa and iodine stains or Gen-Probe but only 50% by ELISA. The direct Giemsa stain provided low sensitivity (25%) and high specificity (100%).

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