Graefe's Arch Clin Exp Ophthalmol (1992)230:411-415
for Clinical and Experimental
Ophthalmology © Springer-Verlag1992
Chlamydia keratoconjunctivitis Determination of Chlamydia trachomatis specific secretory immunoglobulin A in tears by enzyme immunoassay Nico J.F. Buisman 1, Jacobus M. Ossewaarde 2, Marion Rieffe 2, Anton M. van Loon z, and Jan S. Stilma 1 F.C. Donders Institute of Ophthalmology, Academic Hospital, P.O. Box 8500, 3508 GA Utrecht, The Netherlands 2 Laboratory of Virology, National Institute of Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bilthoven, The Netherlands Received July 13, 1991 / Accepted January 3, 1992
Abstract. Determination of Chlamydia trachomatis specific antibodies in serum and tears, isolation of C. trachomatis in cell culture and C. trachomatis antigen detection by direct immunofluorescence test were evaluated for the laboratory diagnosis of Chlamydia trachomatis infection in 30 patients with chronic conjunctivitis. C. trachomatis specific secretory immunoglobulin A (s-IgA) was detected in the tears of all 8 patients with a positive result in culture and the direct immunofluorescence test. In 4 additional patients, the s-IgA assay of tears was also positive. A presumptive clinical diagnosis of Chlamydia conjunctivitis was correlated with the presence of s-IgA in tears. S-IgA was not detected in the serum of any of the patients, indicating that s-IgA is locally produced and not transudated f r o m the serum. For the diagnosis of chronic Chlamydia conjunctivitis, we recommend the determination of s-IgA in tears in addition to a test for Chlamydia detection.
The diagnostic value of tests for determining specific antibodies in tears has been considered controversial because of the possibility of antibody transudation from serum . This is particularly evident in patients with associated genital infection, because of the high antibody titers that m a y be present in their serum. The detection of specific secretory immunoglobulin A (s-IgA), a locally produced Ig in tears, might be a very specific p a r a m e t e r for the diagnosis of chlamydial conjunctivitis . Recently, the detection of herpes simplex virus specific sIgA in tears has been described as a valuable tool for the diagnosis of herpes keratoconjunctivitis . Similarly, the detection of s-IgA in tears has been described for the diagnosis of t r a c h o m a . In this study, we compared the results of the determination of s-IgA in tears of patients with a chronic conjunctivitis with the results of isolation of C. trachomatis in cell culture, and antigen detection by direct immunofluorescence test with clinical criteria for the diagnosis of chlamydial conjunctivitis.
Introduction Conjunctivitis in adults caused by Chlamydia trachomatis is c o m m o n l y associated with genital chlamydial infections . It is m o r e often encountered in sexually active persons and has a chronic course if not treated properly . Recent studies have shown an increase in chlamydial conjunctivitis amongst patients with chronic conjunctivitis [2, 7, 11, 14]. The clinical picture of chlamydial conjunctivitis is often non-specific and hard to distinguish f r o m other bacterial or viral causes. Therefore, a laboratory diagnosis is required [4, 7]. In contrast to acute conjunctivitis, the laboratory diagnosis of chronic conjunctivitis usually presents more difficulties because of a substantial decrease o f viable micro-organisms and inadequate use of topical antibiotics [3, 7]. Isolation of Chlamydia in cell cultures or detection of antigen by direct immunofluorescence test m a y produce false-negative results in chronic conjunctivitis.
Correspondence to: J.M. Ossewaarde
Materials and methods
Patients and controls Thirty patients between 15 and 30 years of age were included in the study. They presented at the Emergency Eye Clinic of the Academic Hospital of Utrecht with symptoms of a conjunctivitis of at least 1 week's duration. No selection was made concerning the presumed etiology. Patients who had used topical antibiotics were first treated with gentamycin eye drops for at least 3 days before specimen sampling. Only serum and tears were collected from a control group consisting of 29 healthy persons in the same age group. The study was carried out with permission of the ethical commission of the Academic Hospital of Utrecht and with the informed consent of patients and controls.
Medical history The sex and age of patients and duration of the conjunctivitis were recorded. Patients were asked about the colour and quantity
412 of the eye discharges, stickiness of the eyelids, crusts, itching, foreign body sensation, pain, photophobia, blurred vision and unilaterality at the start of the disease. In addition, patients were asked for their history of sexually transmitted diseases during the previous 5 years.
(Syva). The elementary bodies in the whole smear were counted. The smear was considered positive if more than 10 elementary bodies were found.
Tear analysis Clinical examination Each patient was examined with a slit lamp. The quality (watery, mucous, mucopurulent or purulent), quantity (no, little, much) and colour (white, yellow, green) of conjunctival discharge were recorded. Conjunctiva were examined for the presence of chemosis, hyperaemia, papillae and follicles of the lower tarsal, upper tarsal and bulbar conjunctiva. The limbus was examined for the presence of follicles and infiltration and the cornea for the presence of keratic infiltrates, scars and pannus formation. A presumptive clinical diagnosis of chlamydial infection was made if at least three of the following four symptoms were present: follicles, infiltration of the limbus, micropannus and keratitis (subepithelial infiltrates). The severity of the conjunctivitis was expressed by the amount of discharge, hyperaemia, papillae, follicles in the upper and lower tarsal conjunctiva and chemosis of the bulbar conjunctiva, all graded from 0 to 3.
Collection of specimens Conjunctival discharge was removed carefully in order to prevent leakage of blood into the conjunctiva. Tears were collected by means of surgical sponges in the inferior fornix as described by van Agtmaal . Conjunctival swabs from the inferior conjunctiva were collected for Chlamydia culture. Swabs from both eyes were placed in one transport vim with 2 ml 4SP (0.4 M sucrose phosphate buffer with 10% fetal calf serum) for culture. Conjunctival smears for direct immunofluorescence test were made from each eye separately. Blood samples were drawn by venipuncture. All specimens were sent to the laboratory within 2 h in a cooler (Coleman 5272).
The presence of IgG, IgM and IgA antibodies to Chlamydia in tears was determined in dilutions of 1:8, 1:16, 1:32 and 1:64 by an indirect immunofluorescence assay using HeLa 229 cells infected with C. trachomatis serovar D. Titers are expressed as negative (1: =64). Secretory IgA was determined in a dilution of 1 : 50 by an enzyme immunoassay using a periodate-treated antigen of C. trachornatis serovar L2 as reported previously  and a monoclonal antibody against the secretory piece of IgA (BioMakor). An adsorption value of more than 1.0 was regarded as highly positive, between 0.2 and 1.0 as positive and less than 0.2 as negative.
Serum analysis The presence of IgG, IgM and IgA antibodies to Chlamydia in serum was determined in dilutions of 1:100, 1:400, 1:1600 and 1:6400 by enzyme immunoassay. Titers are expressed as negative (1 : < 100), highly positive (I : > = 6400 for IgG and 1 : > = 3200 for IgA) or positive. Secretory IgA was determined in a dilution of 1 : 100 by the same method as in tears.
Statistical analysis The data were statistically analyzed with the ~2 test, the Fisher exact test, the H test of Kruskall and Wallis, and the correlation test. Discriminant analysis was used to categorize the patients into 2 groups, 1 with chlamydial conjunctivitis and 1 with nonchlamydial conjunctivitis by different sets of variables. Multivariate analysis of variance was used to determine the significance of this categorization.
Chlamydia culture Specimens were stored in 4SP at - 7 0 ° C. They were thawed and vortexed for 60 s immediately prior to use. One-day-old monolayers of HeLa 229 cells were rinsed twice with 30 gg/ml diethylaminoethanol (DEAE)-dextran in phosphate buffered saline. From each specimen, 0.3 ml was inoculated onto a monolayer in one well of a 24-well microtiter plate and onto a monolayer on a glass cover slip in a flat-bottomed tube. After centrifugation for 60 min at 4800 x g and 25 ° C, the inoculum was replaced by Iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum and antibiotics. After 2 or 3 days' incubation at 37°C and 5% CO~, the monolayers in the microtiter plates were fixed with methanol, stained with fiuorescein-labelled chlamydial-specific monoclonal antibodies (MicroTrak, Syva) and screened ~br the presence of inclusions. The medium of the tubes was replaced by fresh ice cold medium aRer 3 days' incubation, and the tubes were then sonicated in a cup horn for 60 s (Vibra Cell, Sonics and Materials). The tubes were stored at + 4 ° C until inoculation onto a new monolayer on the same day or the next day. One blind passage was performed.
Direct immunofluorescence test Smears were processed immediately. After fixation with methanol, the smears were stained with the MicroTrak direct specimen test
Patients The group of patients with conjunctivitis consisted of 19 m e n a n d 11 w o m e n , r a n g i n g in a g e f r o m 16 to 30 y e a r s ( m e d i a n 25 years). T h e d u r a t i o n o f t h e d i s e a s e w a s 11 d a y s to 10 y e a r s ( m e d i a n 101 days). C o n j u n c t i v i tis w a s f o u n d in a t o t a l o f 53 eyes. T h e c o n j u n c t i v i t i s w a s u n i l a t e r a l in 6 p a t i e n t s . O n e p a t i e n t w a s m o n o c u l u s . O n t h e basis o f c l i n i c a l signs, a p r e s u m p t i v e d i a g n o s i s o f c h l a m y d i a l c o n j u n c t i v i t i s w a s m a d e in 14 p a t i e n t s (Table 1).
Chtamydia culture T h e Chlamydia c u l t u r e w a s p o s i t i v e in 8 p a t i e n t s . T h e c u l t u r e w a s i n i t i a l l y c y t o t o x i c in 3 cases. A f t e r c e n t r i f u g a t i o n f o r 30 r a i n a t 1 2 5 0 0 x g , t h e c y t o t o x i c e f f e c t h a d disappeared, and culture of the deposit of 2 of these samples became positive.
413 Table 1. The relation between medical history and clinical signs of conjunctivitis and Chlamydia culture and the presence of secretory immunoglobulin A (s-IgA) in tears
s-IgA in tears Culture positive Culture negative No s-IgA in tears All patients
Number of patients
Number of patients with clinical Chlamydia diagnosis
Number of patients with STD in history
Mean duration of disease (days)
Mean severity score in most affected eye
12 8 4 18 30
10"** 7 3 4 14
5** 2 3 0 5
116 88* 163"* 123 120
7.4 8.1 6.0 6.7 7.0
STD, Sexually transmitted disease * Difference significant P < 0.05 (Kruskall and Wallis), ** relation significant P