Immunodiagnosis of Adult Chlamydial Conjunctivitis

Immunodiagnosis of Adult Chlamydial Conjunctivitis JOHN D. SHEPPARD, MD,t REGIS P. KOWALSKI, MS,t MICHAEL P. MEYER, MS,2 ANTONIO J. AMORTEGUI, MD,2 MALCOLM SLIFKIN, PhD3

Abstract: This study presents data from a prospective comparison of four currently available diagnostic tests for Chlamydia trachomatis infection. Seventy-six patients clinically suspicious for chlamydial conjunctivitis were all tested with Giemsa stain cytology, direct monoclonal fluorescent antibody (OFA) microscopy, enzyme immunosorbent assay (EIA) for chlamydial antigens, and standard McCoy cell culture. When compared with primary cell culture, diagnostic Giemsa inclusions had a sensitivity and specificity of 43 and 100%, respectively, supportive Giemsa cytology 71 and 67%, the enzyme immunoassay 71 and 97%, and the monoclonal fluorescent antibody 57 and 81 %. Each nonculture method has distinct advantages in terms of cost, technical difficulty, speed, and accuracy, which dictate selection of the most appropriate test for office or laboratory diagnosis of chlamydial conjunctivitis. [Key words: adult inclusion conjunctivitis, Chlamydia trachomatis, direct fluorescent antibody test, enzyme immunoassay, Giemsa cytology.] Ophthalmology 95:434443, 1988

The current epidemic of sexually transmitted diseases is of great concern to both physicians and patients. Numerous organisms afflicting the genital tract also cause ocular disease l ; columnar glandular epithelium is found in the conjunctiva, urethra, and cervix. 2 Genital serotypes of Chlamydia trachomatis are responsible for a plethora of human infections including urethritis and epididymitis in males, cervicitis and salpingitis in females, infant pneumonia, as well as neonatal and adult inclusion conjunctivitis.) Identification of infected patients who first present with ocular chlamydial infection may therefore lead to eradication of extraocular disease in the propositus and their consorts. Originally received: October 20, 1987. Revision accepted: January 8, 1988. Department of Ophthalmology, University of Pittsburgh School of Medicine, The Eye & Ear Hospital, Pittsburgh. 2 Department of Pathology, University of Pittsburgh School of Medicine, MaGee·Womens Hospital, Pittsburgh. 3 Department of Laboratory Medicine, Microbiology Section, Allegheny General Hospital, Pittsburgh. 1

Presented at the American Academy of Ophthalmology Annual Meeting, Dallas, November 1987. The authors have no proprietary interest in the MikroTrak or the Chlamydiazyme tests . Reprint requests to John D. Sheppard, MD, Proctor Foundation, Room S·315, UCSF, San Francisco, CA 94143.

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McCoy cell tissue culture isolation has been the diagnostic standard for chlamydial infections.) This test is expensive, time-consuming, and requires sophisticated tissue culture facilities and highly trained personnel. Cultures are available only through major hospital and university laboratory facilities. Although neonatal chlamydial infection in Western countries can have serious complications, the relatively benign course of adult chlamydial conjunctivitis may prompt empirical ophthalmologic treatment in lieu of a proper diagnostic evaluation. Alternative diagnostic methods which facilitate rapid identification of conjunctival chlamydial infections could justify routine use in an ophthalmic setting. Consequently, this study reports a prospective comparison of four currently available laboratory methods for the diagnosis of adult conjunctival chlamydial infection, including Giemsa stain cytology, direct monoclonal fluorescent antibody (OFA) microscopy, enzyme immunosorbent assay (EIA) for chlamydial antigens, and McCoy cell culture isolation.

SUBJECTS Patients enrolled in this study were recruited from local as well as university-based ophthalmologists and ophthalmology residents. Referral criteria included patients with a chronic or recalcitrant conjunctivitis, an

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Table 1. Study Population No. of Patients

Age (yrs)

Symptoms* (days)

Laboratory Results

Total

M

F

Range

Mean

Range

Mean

Positive culture False-positive Negative

7 15 54

4 8 20

3 7 34

15-35 12-45 14-79

23.6 25.0 32.8

3-90 5-90 2-180

34.1 48.3 77.7

Total

76

32

44

12-79

30.4

2-180

67.9

* Maximum days recorded was 180. Several patients in the negative group mentioned symptoms that lasted longer than 1 year.

examination highly consistent with chlamydial inclusion conjunctivitis, a known extraocular chlamydial infection, or contact with a person known to have a chlamydial infection. The laboratory was not used to screen all cases of conjunctivitis. Findings on initial examination included pseudoptosis, mucopurulent discharge, papillary conjunctivitis with and without follicles, limbal swelling, and superficial punctate keratitis. All patients included had not received oral antibiotics for 72 hours or topical antibiotics for 48 hours before testing. Patients were followed for at least 6 weeks after testing. Seven patients with incomplete test results, contaminated cultures, or inadequate follow-up were excluded from analysis. Only one of these seven patients had any positive results, which was a single, positive DFA test. Neonatal patients were not included in this analysis. All 76 patients reported in this study agreed to the terms of informed consent as defined by the Eye & Ear Hospital Institutional Review Board. Parents signed for patients under 18 years of age. There were 32 males and 44 females tested (mean age, 30.4 years) (Table 1). The average duration of symptoms before testing was 67.9 days, with a maximum recorded duration of 180 days.

SPECIMEN COLLECfION All specimens were obtained under standardized conditions by the same ophthalmic microbiology technician (RPK) in the Campbell Ophthalmic Laboratory, Department of Ophthalmology, University of Pittsburgh. Patients first received routine lid and conjunctival bacterial cultures, obtained with sterile cotton-tipped applicators moistened with trypticase soy broth. Patients were then given one drop of proparacaine in each eye followed by scrapings of upper and lower tarsi bilaterally, first for Giemsa cytology and then for DFA. Giemsa slides were heat fixed and processed immediately using freshly prepared reagent (Harleco stain, Gibbstown, NJ). Slides for DFA were fixed immediately with cold, water-free acetone and stored at 2°C. Thereafter, vigorous inferior forniceal swabs were taken from both eyes using two cotton-tipped swabs on plastic shafts. In patients with unilateral conjunctivitis, scrapings and swabs were first obtained from the unaffected eye to avoid contralateral inoculation. One swab speci-

men was placed in 1.5 ml of chlamydial transport media and stored at 2°C for culture testing. Culture transport media was composed of 0.1 M sucrose in 0.02 phosphate buffer (pH 7.2) supplemented with 10% fetal bovine serum prescreened for anti-chlamydial antibodies, 0.125 mg/ml vancomycin, 0.05 mg/ml streptomycin, 25 U/ml nystatin, and 0.01 mg/ml amphotericin B. The EIA transport tubes supplied by the manufacturer were hit briskly with a fingernail in order to saturate the specimen tip with the 0.1 cc of transport media contained within. Direct monoclonal fluorescent antibody was performed within 7 days and EIA within 72 hours of specimen procurement, as recommended by the manufacturers. Bacterial cultures and Giemsa cytology were performed at the Eye & Ear Hospital, EIA and McCoy cell cultures were performed by MaGee-Womens Hospital, and DFA tests were performed at Allegheny General Hospital. Specimens were transported at 2° to 8°C. Each laboratory was masked to the results of other diagnostic tests.

LABORATORY METHODS Lid and conjunctival culture specimens were placed on blood, mannitol, chocolate, and Saboraud's agar, and into liquid thioglycollate media. Blood and chocolate agar plates were incubated at 37°C aerobically, mannitol and thioglycollate media were incubated at 37°C in 5% CO 2 anerobically, and Saboraud's media were incubated at room temperature in room air. All specimens were read at 24 and 48 hours, whereas fungal and anerobic media were incubated for a total of 7 days before final reading. Giemsa specimens were examined for intracytoplasmic inclusions (Halberstaedter-Prowazek), bacterial organisms, and inflammatory cell responses. Characteristic mixed polymorphonuclear leukocyte and lymphocyte infiltrates, with or without follicle cells, plasma cells or Leber cells, were considered supportive cytology.4 More homogeneous cellular responses, those considered to be atypical of chlamydial infection, or those suggestive of viral, bacterial, or allergic disease, were considered to be nonsupportive. Only the presence of intraepithelial cytoplasmic inclusions was considered to be a diagnostic Giemsa test. 435

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Direct monoclonal fluorescent antibody prefixed, 8-mm well slides were treated with 30 ~l of Evan's blue counterstain and fluorescein-conjugated monoclonal antibody to the major outer membrane protein of C. trachomatis (Mikrotrak, Syva, Palo Alto, CA), incubated for 15 minutes at room temperature, rinsed with sterile water, washed again, and air dried. Slides were scanned at Xl00 and read at x640 magnification under oil immersion by Zeiss fluorescent microscopy (Carl Zeiss, Oberkochen, FRG). A positive DFA required the identification of five or more characteristic apple-green fluorescent elementary bodies per slide, read by two independent experienced observers masked to one another's results. Specimens were considered inadequate if there were less than 20 epithelial cells per high power field or less than 200 epithelial cells per slide. Enzyme immunosorbent assay testing was performed with commercially available kits (Chlamydiazyme, Abbott Laboratories, North Chicago, IL). After allowing specimens and reagents to reach room temperature, 1 ml of buffer was added to each tube. After 10 minutes at room temperature, the tubes were mixed in a Model 2600 Multivortex (Scientific Manufacturing Industries, Emeryville, CA) at a setting of 4 for three cycles of 15 seconds each. The swabs were vigorously expressed and then discarded. Next, 200 ~l of each sample as well as positive and negative controls were pipetted individually into 9-mm wells of a 20-well reaction tray. A treated polystyrene bead was added to each well. The tray was covered, tapped gently to remove air bubbles, and incubated for 30 minutes in a 37°C water bath. After this initial incubation, all beads were washed three times with distilled water using an Abbott Pentawash. Then 200 ~l of a solution of polyclonal rabbit antichlamydial antibodies were added to each well. The tray was covered, tapped, and incubated for 60 minutes in the 37°C water bath. Next, the beads were washed and 200 ~l of horseradish peroxidase-conjugated goat antirabbit immunoglobulin were pipetted into each reaction well. Once again, the tray was sealed and incubated as above. During the last 10 minutes of this third incubation, a fresh 2.56 mg/ml substrate solution of o-Phenylenediamine-2HCI in citrate-phosphate buffer containing 0.2% H20 2 was prepared. The beads were washed once more, transferred to plastic assay tubes, and 300 ttl of the substrate solution were added to each specimen tube, to the control tubes, and also to two empty tubes. All tubes were then held in the dark for 30 minutes at room temperature. To stop the substrate reaction, 1 ml of 1 N H 2S04 was added to each tube. The tubes were briefly vortexed and read at 492 nm in a spectrophotometer (Quantum II, Abbott Laboratories, North Chicago, IL). The tubes containing only one substrate solution and acid were used as blanks. Specimens were reported as positive if the absorbance reading was equal to or above a cutoff value calculated by adding 0.100 to the average absorbance reading for the negative controls. McCoy cell culture specimens were either kept at 2°C and processed within 24 hours, or frozen at -80 0 and processed within 7 days. The swabs were expressed and 436

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1 ml of an inoculation medium consisting of Eagle's minimum essential medium with Hanks' salts, L-glutamine, and supplemented with 10% fetal bovine serum, 5% additional glucose, 0.05 mg/ml streptomycin, 0.1 mg/ml vancomycin, 0.005 mg/ml amphotericin B, 2.5% HEPES buffer (1 M), and 37.5 mg/ml NaHC0 3 was added to each specimen. At least two confluent McCoy cell monolayers grown on 13-mm glass coverslips in 1 dram shell vials were inoculated with 0.2 to 0.5 ml of the chlamydial transport medium-inoculation medium mixture after aspirating off the original McCoy cell growth medium. The inoculated monolayers were centrifuged at 2800 X G for 60 minutes at 35°C. Afterward, the inoculum was removed and 1.5 ml of inoculation medium containing 1 ~g/ml of cycloheximide was added to each vial. After 65 to 72 hours of incubation at 37°C, both monolayers were stained for chlamydial inclusions using immunofluorescent monoclonal antibody (Syva, Palo Alto, CA). Cultures were considered positive if one or more intracytoplasmic inclusions were observed on either coverslip.

STATISTICAL ANALYSIS Sensitivity and specificity for the Giemsa, OFA, and EIA tests were calculated using McCoy cell culture results as a standard for positivity in disease and negativity in health. The predictive value positive (or true-positive results divided by the sum of true-positive and false-positive results) indicates the probability that a positive test will coincide with a positive culture. Similarly, predictive value negative is the quotient of true negatives divided by the sum of true- and false-negative results. Test efficiency is the sum of true-positives plus true-negatives, divided by the total number of tests performed. The kappa statisticS was used to indicate the degree of agreement among the four tests. Different tests are in agreement when both are positive or both are negative for a given specimen. Highly concordant test results have a kappa approaching 1.00. This statistic enables simultaneous comparison of numerous tests and provides an estimate of test efficiency. It is a useful adjunct to standard sensitivity and specificity evaluations, although subject to the same limits of sample size.

RESULTS A total of 22 patients had at least one positive diagnostic chlamydial test (Table 1). Ten were female and 12 were male. Fifteen patients had a false-positive EIA or a false-positive DFA test. The mean age and symptom duration figures for the false-positive group fall between the true-positive and the negative groups. Fifty-four patients had negative diagnostic chlamydial tests, of which 34 were female and 20 male. The mean age ofthis group was 32.8 years and the mean duration of symptoms pretesting was 77.7 days. Patients with positive diagnostic tests were younger and had a shorter duration of

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Table 2. Positive Diagnostic Chlamydial Tests No. of Patients 3 2 1 1 2 13

EIA

DFA

McCoy

Giemsa

+ +

+

+ + + +

Inclusions

7

3

+

Total 22

7

+ + 17

EIA = enzyme immunosorbent assay; DFA = direct monoclonal fluorescent antibody.

ocular symptoms before testing than those with negative tests. Three Giemsa smears were positive for inclusions and therefore diagnostic (Table 2). All three, however, also showed positive EIA, DFA, and culture tests. There was a total of 7 positive EIA tests, 17 positive DFA tests, and 7 positive McCoy cell cultures. In Table 3, sensitivity, specificity, and predictive values are given for Giemsa, DF A, and EIA results compared with cell culture. The best correlation with McCoy cell culture was obtained with EIA testing, which had the highest kappa statistic (Table 4). The Giemsa, EIA, and DFA tests did not correlate very well with each other.

Giemsa cytology was correctly supportive or diagnostic in five of seven culture-positive patients, and correctly nonsupportive in 67% of culture-negative patients, with an overall accuracy of 67% (Table 5). Although supportive cytology is not considered to be diagnostic, Giemsa cytology effectively screened two thirds of the study patients, compared with 79% by DFA, 95% by EIA, and 95% by Giemsa inclusions. Eighteen patients with positive diagnostic chlamydial tests were treated with oral tetracycline (250 mg orally four times daily) for 3 weeks (Table 6). All seven culture-positive patients responded to treatment. Nine of 11 patients with false-positive results responded completely to therapy, one improved but could no longer tolerate soft contact lenses, and one did not improve requiring surgery for superior limbic keratoconjunctivitis. Four of the false-positive patients, all by DFA alone, were not treated by their referring physicians. Two of these remained symptomatic and two resolved spontaneously. A positive DFA test predicted 13 favorable responses to appropriate antibiotic therapy, whereas the EIA and culture test predicted seven each. The two treated patients who did not completely respond as well as the two with spontaneous resolution of symptoms were all positive only for the DFA test. Of the two initially untreated false DFA-positive patients who remained symptomatic, one patient received tetracycline and resolved, whereas the other responded to topical silver nitrate for superior limbic keratoconjunctivitis

Table 3. Sensitivity and Specificity' McCoy Cell Culture Test

Result

Positive

Negative

DiagnostiC Giemsa inclusions Positive Negative

3 4

0 69

Supportive Giemsa cytologyt Positive Negative

5 2

23 46

EIA Positive Negative

5 2

2 67

DFA Positive Negative

4 3

13 56

Accuracy Sensitivity Specificity Predictive value positive Predictive value negative Efficiency

0.429 1.000 1.000 0.945 0.947

Sensitivity Specificity Predictive value positive Predictive value negative Efficiency

0.714 0.667 0.179 0.958 0.671

Sensitivity Specificity Predictive value positive Predictive value negative Efficiency

0.714 0.971 0.714 0.971 0.947

Sensitivity Specificity Predictive value positive Predictive value negative Efficiency

0.571 0.812 0.235 0.949 0.789

EIA = enzyme immunosorbent assay; DFA = direct monoclonal fluorescent antibody . • Data are based on primary McCoy cell culture results as the standard. t Giemsa cytology with either inclusions or supportive cytology is considered positive. Supportive Giemsa cytology without inclusions is not diagnostic.

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Table 4. Kappa Statistic: Inter-test Correlation Supportive Giemsa Cytology

Test Diagnostic Giemsa inclusions Supportive Giemsa cytology EIA DFA

0.13

EIA = enzyme immunosorbent assay; DFA rescent antibody.

=

EIA

DFA

0.58 0.23

0.25 0.23 0.14

McCoy Culture 0.58 0.16 0.69 0.23

direct monoclonal fluo-

Giemsa Reading

Positive cell culture Negative cell culture Total

3 2 2 15 39

Accuracy

Inclusions Supportive Nonsupportive Supportive Nonsupportive

71% (5/7) 67% (46/69)

76

67% (51/76)

, Giemsa cytology with inclusions considered diagnostic. Mixed polymorphonuclear leukocyte and lymphocyte response with or without plasma cells, follicle cells, or Leber cells is considered supportive. Homogeneous response, bacteria, eosinophils, or picture more typical of other diseases considered nonsupportive. Table 6. Initial Clinical Outcome

Test Positive McCoy cell culture (n = 7) False-positive chlamydial test (n = 15)t

No. Patients Treated

7* 11

4

Outcome 7 asymptomatic 9 asymptomatict 1 improved but unable to use SCLs no improvement, conjunctival resection for SLK 2 still symptomatic 2 spontaneous resolution

SCLs = soft contact lenses; SLK = superior limbic keratitis. * All treated patients were given oral tetracycline (250 mg orally four times daily) for 3 weeks, with or without adjunctive topical medications. t Thirteen patients had a false-positive direct fluorescent antibody and two had a false-positive EIA. t Includes seven direct monoclonal fluorescent antibody-positive and two enzyme immunosorbent assay-positive patients.

(Table 7). All patients with positive EIA, cultures, or Giemsa inclusions responded when given antibiotic therapy. Fifty-four patients referred to this study did not have any positive diagnostic chlamydial tests. Blepharitis, allergic conjunctivitis, and soft contact lens allergy were the most common final diagnoses made by referring 438

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Table 7. Final Diagnosis of False-positive Tests (n

Table 5. Giemsa Cytology' Patient Group

•

No. of Patients

=

15)

Final Diagnosis Presumed AIC with good tetracycline response' Soft contact lens allergy' Allergic conjunctivitis Superior limbic keratoconjunctivitist Bacterial conjunctivitis

9 3

1 1 1

AIC = adult inclusion conjunctivitis. • Of the two patients with a false-positive enzyme imunosorbent assay, one had a final diagnosis of AIC, the other, soft contact lens allergy. t Initially untreated direct monoclonal fluorescent antibody-positive patient responded to topical silver nitrate but never received tetracycline. Table 8. Final Diagnosis· (n No. of Patients 11

9 9 5 4 4 4

3 3 3 2 2 1 1 1 1

1

1

= 54) Negative Chlamydial Tests Final Diagnosis

Blepharitis Allergic conjunctivitis Soft lens allergy Rosacea Keratitis sicca Possible viral conjunctivitis Culture-positive viral conjunctivitis Medicamentosa Corneal erosion Bacterial conjunctivitis Old trachoma Corneal dystrophy Molluscum contagiosum Benign lymphoid hyperplasia Pterygium Superior limbic keratitis Floppy eyelid syndrome Nodular episcleritis

• Total number of alternate diagnoses after negative testing exceeds the total number of patients (54) since several patients had more than one final diagnosis.

physicians once inclusion conjunctivitis had been ruled out (Table 8). Academic cornea/external disease specialists, private fellowship-trained cornea specialists, general ophthalmologists, and ophthalmology residents referred patients to this study (Table 9). General ophthalmologists most frequently referred patients suspicious for chlamydial conjunctivitis who actually had one or more positive diagnostic tests (31 %), whereas residents most frequently referred patients thought to have chlamydial disease who were actually culture-positive (13%).

DISCUSSION C. trachomatis is a gram-negative, nonmotile intracellular prokaryotic organism. This bacterium is an obligate energy parasite incapable of synthesizing its own adenosine triphosphate. C. trachomatis is the causative

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Table 9. Physician Study Referrals; Accuracy of Initial Clinical Impression

Positive culture Negative culture

% positive Any positive test* All tests negative

% positive

Academic (n = 21)

Corneal (n = 16)

General (n = 16)

Resident (n = 23)

(n = 7) (n = 69)

1 20

1 15

2 14

3 20

10

5

6

8

13

(n = 21) (n = 55)

6 15

4 12

5 11

6 17

28

29

25

31

26

* This includes positive enzyme immunoassay or a positive direct fluorescent antibody test. One patient who had a positive culture and no other positive tests has been excluded. This would simulate screening with the immunodiagnostic tests alone.

organism of the most common sexually transmitted disease in the United States6 as well as the leading cause of preventable blindness worldwide. 7 Adult inclusion conjunctivitis and chlamydial ophthalmia neonatorum are caused by the same chlamydial serotypes found in genital infections.8 Both syndromes are invariably acquired by contact with infective genital tract discharge,9 through sexual intimacy, hand to eye contact, or via the birth canal. In adults, inclusion conjunctivitis presents as an acute follicular inflammation and mucopurulent discharge without systemic manifestations. The incubation period from volunteer studies is 4 to 12 days. The disease is most prevalent in young adults who have acquired a new sexual partner in the 2 months preceding the onset of symptoms. Epithelial keratitis, most prominent superiorly, may develop during the second week. Further progress may include marginal and central infiltrates, subepithelial opacities, limbal swelling, and superficial vascularization. \0 The course is usually benign and self-limited, but may become chronic with conjunctival scarring and pannus formation. Chlamydial infection is an extremely common cause of infectious ophthalmia neonatorum in the United States. II Neonatal chlamydial conjunctivitis may require prolonged oral erythromycin therapy and may be complicated by chlamydial pneumonitis. 12 Diagnosis of ocular chlamydial infection is also epidemiologically important in the identification of genital sexually transmitted diseases. 13-15 Evaluation of new laboratory tests for the diagnosis of chlamydial infection can be frustrating due to the lack of a true gold standard. Falsely negative McCoy cell cultures may be a result of prior antibiotic therapy.1 6 The effects of topical anesthetics on chlamydial viability are unknown. Higher culture-positive yields may have been obtained in this study by waiting a full week off antibiotic treatment before testing, since numerous study patients had been treated before referral. This would have been logistically difficult. Secondary passage subcultures also may have improved the yield of positive cultures,17 although this adds further to laboratory expense. Cell culture techniques rely on chlamydial infectivity and viability, which are dependent on the hospital-

ity of mucous membranes and host secretions, the integrity of transport media, avoidance offreeze-thaw cycles before culturing, proper and rapid laboratory processing, and the absence of antibiotics with in vitro antichlamydial activity.1 8 Nevertheless, had only a few apparently false-positive DFA or EIA tests been corroborated by a newly positive second passage cell culture, the sensitivity of these two nonculture tests may have improved significantly. Perhaps the spread of data presented in Table 2 would have been reduced had a second passage been performed. Subsequent trials using these tests should employ second passage culture. Immunodiagnostic techniques, on the other hand, do not depend on live organisms. They rely instead on highly specific, labeled antibodies to chlamydial surface antigens. The EIA is an indirect test using primary polyclonal antichlamydial antibodies and secondary enzyme-conjugated polyclonal antibodies capable of colorimetric change when incubated with the appropriate substrate. The DFA test directly labels individual chlamydial elementary bodies with a monoclonal antibody to the chlamydial species specific outer membrane determinane 9 conjugated to fluorescein isothiocyanate. Neither test is currently approved for the diagnosis of adult ophthalmic disease, although both are now approved for evaluation of neonatal conjunctivitis. The EIA showed the best correlation with McCoy cell culture (Table 4). This test has been shown to be as sensitive as first-pass cell culture in detecting genital chlamydial infections.20 Although the EIA and culture tests were performed in the same facility for this study, arguments that their agreement may have been artificially enhanced are unfounded due to the strictly objective optical density readout in the EIA. This study, to our knowledge, is the first to describe the EIA in the diagnosis of adult inclusion conjunctivitis. The DFA had the highest number of positive tests (Table 2) and predicted the greatest number of positive responses to antibiotic therapy (Table 6). Blepharitis, bacterial disease, and rosacea blepharoconjunctivitis also may respond to tetracycline treatment. This OFA is useful in the diagnosis of genital chlamydial infections,21 neonatal conjunctivitis, \0 trachoma,22 and experimental 439

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ocular infections. 23 The DFA test can be used successfully in the diagnosis of adult inclusion conjunctivitis,24 although false-positives also have been reported in other studies. 25 ,26 The large number of positives in this study may be due to the fact that the DFA scraping was taken before the EIA and culture specimen . On the other hand, prior scraping may have liberated free elementary bodies from essentially mature inclusions to increase the EIA and culture yields. Although the DFA may someday prove to be inherently more sensitive than culture, the possibility of false-positives must be considered especially when confronting a patient with the diagnosis of a sexually transmitted disease. The MicroTrak test has been used to tentatively identify asymptomatic carriers of ocular chlamydia in patients attending a sexually transmitted disease clinic,27 and to corroborate Giemsa inclusions in a symptomatic patient with recalcitrant superior limbic disease?8 Unfortunately, conjunctival McCoy cell cultures were not done in either report. Isolated DFA positivity in our patient requiring surgery for superior limbic keratitis as well as the patient with spontaneous symptomatic resolution (Table 6) leaves a diagnosis of chlamydial disease somewhat suspect. Four of 13 patients with false-positive DFA results were not given a final clinical diagnosis consistent with chlamydial disease by their referring physician (Table 7). Had second passage cultures been used, DF A specificity and sensitivity may have been improved. Specificity improved only slightly when data were reviewed using ten rather than five elementary bodies per slide for a positive test. Generally speaking, DFA slides showed either a great abundance or no elementary bodies whatsoever. The DFA appears to be more sensitive with methanol rather than acetone fixation, according to recent recommendations from the manufacturer. Although the manufacturer suggests the use of a Dacron swab, all of the specimens obtained in this study with a platinum spatula had an adequate number of epithelial cells. Attention to high-quality fluorescent optics is essential to allow optimal visualization and differentiation of fluorescein debris from labeled elementary bodies, although the instrument used in this study complies with instructions from the manufacturer. Finally, the experience of any observer with any new test may influence subjective interpretation. Whether or not the 15 false-positive tests represent failure of isolation in cell culture is uncertain. One of the two false-positive EIA patients and 8 of the 13 false-positive DF A patients responded to oral tetracycline and received a final diagnosis of presumed adult inclusion conjunctivitis (Table 7). One of the two false-positive EIA patients had supportive Giemsa cytology, the other did not. Seven of the 13 false-positive DFA patients had supportive Giemsa cytology. In these 15 false-positive patients, the supportive cytology did not correlate at all with the treatment outcome. Perhaps more specific diagnostic technology independent of chlamydial organism viability, such as the DNA probe, will be necessary to finally determine the ultimate use of nonculture diagnostic tests. False-positive DFA or EIA tests may, in 440

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Table 10. Cost-effectiveness Comparison

Test

Test

Cost

True Positive Missed

Confirmation

Screening Cost

Total Costs

Culture DFA EIA Giemsa

$85 $25 $47 $15

76 76 76 76

None Culture Culture Culture

None $85 X 17 $85 X 7 $85 X 25

0 3 2 2

$6460 $3345 $4167 $3265

DFA EIA

$36 X 76 $36 X 76

Culture Culture

$85 X 17 $85 X 7

3 2

$4181 $3331

X X X X

DFA = direct monoclonal fluorescent antibody; EIA sorbent assay.

= enzyme immuno-

fact, represent persistence of chlamydial antigens in the absence of replicative viability. The Giemsa stain, although not highly sensitive in this study, proved useful as a screening device. Supportive cytology showed respectable sensitivity and specificity (Table 3), and an overall efficiency of 67% (Table 5). Giemsa inclusions are found far more frequently in neonatal than in adult chlamydial ocular disease. 29 The use of Giemsa stain screening of patients with suspected adult inclusion conjunctivitis has been noted in other studies. 30 Strict interpretation of conjunctival exudates must be made with reservation, however, when implicating chlamydial etiology.31 Laboratory charges at the time of this study were approximately $15 for the Giemsa stain, $47 for the EIA, $25 for the DFA, and $85 for McCoy cell culture. Giemsa staining takes at least 45 minutes, the EIA 3 hours, the DFA test about 45 minutes, and single-passage cell culture 72 hours. Both the Giemsa and the DFA could be accurately interpreted by a willing ophthalmologist, assisted in specimen preparation by trained office staff. Both DFA and Giemsa evaluation offer the distinct advantage that the specimens can be assessed for adequacy. False-negative results can follow the procurement of insufficient conjunctival material from squeamish patients by hesitant physicians or technicians. A Kimura platinum spatula was used to obtain the DFA and Giemsa specimens in this study. Direct monoclonal fluorescent antibody specimens also can be procured with a Dacron or cotton-tipped swab, which theoretically might decrease their yield. This might explain the higher specificity and lower sensitivity for MicroTrak in other studies. 2o Ophthalmologists at all levels of training had difficulty in making a diagnosis of laboratory-confirmed adult inclusion conjunctivitis based on clinical and historic findings alone, since the overall rate for test positivity was only 28%, and 10% for culture positivity. Normal referral patterns may have been artificially altered after professionally disseminated publicity regarding this study caused an increased awareness of chlamydial disease in the local ophthalmic community. Although such an awareness is desirable, more stringent selection of patients for testing would have reduced culture costs in a more routine setting. Nevertheless, the cost of

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screening with immunodiagnostic tests leading to additional follow-up cultures for positive results is far less than the cost of screening all 76 study patients with culture alone (Table 10). According to laboratory fees charged at the time of this study, the Giemsa stain is the most cost-effective screening test, followed by the DFA. If laboratory fees for the DFA and EIA are averaged to $36 each, then the EIA would be more cost-effective than the DFA. There are, of course, disadvantages in bringing patients back for a second test, so conjunctival swabs in culture transport media could be saved and frozen after the initial visit pending results of a Giemsa, DFA, or EIA screen.

Giemsa stain, although rapid and perfect in specificity, has limited sensitivity and requires skilled interpretation. Giemsa stains do allow for assessment of specimen adequacy and permit general office screening for a host of external ocular diseases. Only a standard light microscope is required, although daily preparation of fresh Giemsa stain is considered essential by many experienced observers. The newer immunodiagnostic tests do show considerable promise in the diagnosis of adult inclusion conjunctivitis in this study. The Syva MicroTrak DFA and the Abbott Chlamydiazyme EIA are both currently available for evaluation. Further assessment of their capabilities and limitations are in order before final recommendations can be approved for their general use.

CONCLUSION

ACKNOWLEDGMENTS

Each of these tests offers distinct advantages when considering routine application in the office or the ophthalmic laboratory. The Giemsa stain remains the most cost-effective general screening test for external ocular diseases. When chlamydial infection must be definitively or medicolegally diagnosed, cell culture remains the test of choice. Cell culture is the established standard even though there are disadvantages in terms of cost, time, availability of tissue culture facilities, and the inability to assess specimen adequacy. When carefully evaluating selected high-risk groups for chlamydial disease, DFA or EIA tests offer advantages over the culture method as well as an opportunity to provide cost-effective screening. These tests also are the only alternative in remote locales or Third World countries. Direct monoclonal fluorescent antibody and EIA are relatively inexpensive, reliable, rapid, immunodiagnostic techniques that may become more and more a part of routine ophthalmic laboratory testing. Only 10% of study subjects actually had a positive culture, even though patients highly suspicious for chlamydial disease were solicited from referring physicians. Thus, more stringent patient selection can render laboratory testing more cost-effective. Conditions most commonly mistaken for adult inclusion conjunctivitis were blepharitis, allergic conjunctivitis, soft contact lens allergy, and rosacea blepharoconjunctivitis. The EIA showed superior sensitivity and specificity in this study, and permits strictly objective interpretation of results, although specimen adequacy cannot be assessed. The Chlamydiazyme EIA has not been adapted previously to adult inclusion conjunctivitis. The DFA test, although not optimized in this study, does offer the advantage of assessment of specimen adequacy and the availability of numerous previous ophthalmic studies for comparison. Reading the DFA test is subject to the vagaries of subjective interpretation, reader experience, and fluorescence microscope optical quality. Since the EIA requires a spectrophotometer, the DFA a fluorescence microscope, and both tests require immunologic reagents with a limited shelf life, their current use may be limited to high volume laboratory facilities. The

Reagents and equipment for some of the tests performed in this study were provided through the generosity of Abbott Pharmeceuticais, Chicago IL, and Syva Company, Palo Alto, CA.

REFERENCES 1. Dawson CR, Whitcher JP, Lyon C, Schachter J. Response to treatment in ocular chlamydial infections: analogies with nongonococcal urethritis. In: Hobson D, Holmes KK, eds. Nongonococcal Urethritis and Related Infections. Washington, DC: American Society for Microbiology, 1977; 135-9. 2. Naib ZM. Cytology of TRIC agent infection of the eye of newbom infants and their mothers' genital tracts. Acta Cyto11970; 14:390. 3. Schachter JS, Dawson CR. Human Chlamydial Infections. Littleton, Mass: PSG Publishing Co, 1978; 181-219. 4. Yoneda C, Dawson CR, Daghfous T, et al. Cytology as a guide to the presence of chlamydial inclusions in Giemsa-stained conjunctival smears in severe endemic trachoma. Br J Ophthalmol 1975; 59:116-24. 5. Steel RGD, Torrie JH. Principles and Procedures of Statistics: A Biometrical Approach, 2nd ed. New York: McGraw-Hili, 1980. 6. Thompson SE, Washington AE. Epidemiology of sexually transmitted Chlamydia trachomatis infections. Epidemiol Rev 1983; 5:96-123. 7. WHO Programme Advisory Group on the Prevention of Blindness. Data on blindness throughout the world. WHO Chron 1979; 33:27583. 8. Grayston JT, Wang Sop. New knowledge of Chlamydiae and the diseases they cause. J Infect Dis 1975; 132:87-105. 9. Schachter J, Rose L, Meyer KF. The venereal nature of inclusion conjunctivitis. Am J Epidemiol 1967; 85:445-452. 10. Dawson CR. Follicular conjunctivitis. In: Duane TD, Jaeger EA, eds. Clinical Ophthalmology. Philadelphia: Harper & Row, 1985; vol. 4, chapter 7. 11. Rapoza PA, Quinn TC, Kiessling LA, Taylor HR. Epidemiology of neonatal conjunctivitis. Ophthalmology 1986; 93:456-61. 12. Rapoza PA, Kiessling LA, Quinn TC, Taylor HR. Ophthalmia neonatorum: an epidemiologic survey and diagnostic study. ARVO Abstracts. Invest Ophthalmol Vis Sci 1985; 26(Suppl):272. 13. Schachter J. Chlamydial infections: (first of three parts). N Engl J Med 1978; 298:428-35. 14. Schachter J. Chlamydial infections: (second of three parts). N Engl J Med 1978; 298:490-5. 15. Schachter J. Chlamydial infections: (third of three parts). N Engl J Med 1978; 298:540-9.

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16. Westrom L, MArdh PA. Chlamydial salpingitis. Br Med Bull 1983; 39:145-50. 17. Amortegui AJ, Meyer MP. A nonculture test for identification of Chlamydia trachomatis. J Reprod Med 1985; 30:279-83. 18. Blackman HJ, Yoneda C, Dawson CR, Schachter J. Antibiotic susceptibility of Chlamydia trachomatis. Antimicrob Agents Chemother 1977; 12:673-7. 19. Stephens RS, Tam MR, Kuo C-C, Nowinski RC. Monoclonal antibodies to Chlamydial trachomatis: antibody specificities and antigen characterization. J Immunol 1982; 128: 1083-89. 20. Amortegui AJ , Meyer MP. Enzyme immunoassay for detection of Chlamydia trachomatis from the cervix. Obstet Gynecol 1985; 65:523-6. 21. Tam MR, Stamm WE, Handsfield HH, et al. Culture-independent diagnosis of Chlamydia trachomatis using monoclonal antibodies. N Engl J Med 1984; 310:1146-50. 22. Wilson MC, Millan-Velasco F, Tielsch JM, Taylor HR. Direct-smear fluorescent antibody cytology as a field diagnostic tool for trachoma. Arch Ophthalmol1986; 104:688-90. 23. Taylor HR, Agarwala N, Johnson SL. Detection of experimental Chlamydial trachomatis eye infection in conjunctival smears and in tissue culture by the use of fluorescein-conjugated monoclonal antibody. J Clin MicrobioI1984; 20:391-5.

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24. Taylor HR, Rapoza PA, Kiessling LA, Quinn TC. Rapid detection of Chlamydia trachomatis with monoclonal antibodies. Lancet 1984; 2:38. 25. Ugland DN, Jones DB, Wilhelmus KR, Osato MS. Diagnosis of adult chlamydial conjunctivitis by use of fluorescein-conjugated monoclonal antibody. ARVO Abstracts. Invest Ophthalmol Vis Sci 1985; 26(Suppl):272. 26. Bialasiewicz AA, Jahn GJ. Evaluation of diagnostic tools for adult chlamydial keratoconjunctivitis. Ophthalmology 1987; 94:532-7. 27. Insler MS, Anderson AB , Murray MA. Latent oculogenital infection with Chlamydia trachomatis. Ophthalmology 1987; 94:27-9. 28. Seedor JA, Droste PJ, Perry HD. Chlamydial infection producing keratoconjunctivitis of the comeoscleral limbus. Am J Ophthalmol 1986; 102:798-800. 29. Schachter J, Dawson CR. Comparative efficiency of various diagnostic methods for chlamydial infection. In: Hobson D, Holmes KK, eds. Nongonococcal Urethritis and Related Infections. Washington, DC: American Society for Microbiology, 1977; 337-41. 30. Wilhelmus KR, Robinson NM, Tredici LL, Jones DB. Conjunctival cytology of adult chlamydial conjunctivitis. Arch Ophthalmol 1986; 104:691-3. 31. Stenson S, Newman R, Fedukowicz H. Laboratory studies in acute conjunctivitis. Arch Ophthalmol 1982; 100:1275-7.

Discussion by Chandler R. Dawson, MD These studies by Dr. Wiley and by Dr. Sheppard and their respective associates deal with the problem oflaboratory diagnosis of nonbacterial conjunctivitis. The first question the practicing ophthalmologist must ask is: How useful are these tests for the management of patients? For chlamydial infection, the answer is clear-cut: A positive diagnosis of Chlamydia trachomatis infection is an indication for 2 to 3 weeks therapy with oral tetracycline, doxycycline, or erythromycin. For adult inclusion conjunctivitis, a positive chlamydial test also indicates that the patient's sexual consorts should be treated at the same time because chlamydial disease of the genital tract is an important cause of morbidity, even sterility in women. For adenovirus infections, the immediate treatment benefits of diagnostic tests for adenovirus of the eye are less apparent. Nevertheless, a positive test is useful. It indicates that the condition is self-limited and will not recur. It also alerts the physician to possible appearance of subepithelial opacities as the conjunctivitis clears. The tests for adenovirus infection would be even more useful if combined with a similar test for herpes simplex infection which accounts for about 10% of acute follicular conjunctivitis. The problem for the practicing ophthalmologist is when to use these tests: What kinds of patients should be tested with these laboratory procedures? From Dr. Wiley's presentation, it is apparent that tests for adenovirus infection are only useful in acute conditions, particularly acute follicular conjunctivitis. These conditions include: adenovirus (Beal's) follicular conjunctivitis (pharyngoconjunctival fever, epidemic keratoconjunctivitis), acute chla-

From the Francis I. Proctor Foundation for Research in Ophthalmology, University of California, San Francisco.

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mydial conjunctivitis with genital C trachomatis serovars, herpetic follicular conjunctivitis, Newcastle disease virus conjunctivitis, and acute hemorrhagic conjunctivitis. In its early stages, however, adenovirus conjunctivitis may not present with characteristic follicles but with an acute papillary conjunctivitis often with watery discharge and chemosis which may be confused with an acute allergic response. Although there are two reports in the literature of adenovirus isolations from cases of chronic conjunctivitis, this virus has never been recovered from the eye after second week of onset. Thus, tests for adenovirus infection are only useful in the acute forms of non bacterial conjunctivitis. In contrast, C trachomatis infections of the eye cause a chronic follicular conjunctivitis which may persist for many months. In its acute presentation, chlamydial conjunctivitis may resemble the adenovirus infections for the first 2 weeks, although it is often accompanied by some muco-pus and unlike the adenovirus infections rarely produces membranous conjunctivitis. There are a large number of conditions which present or may be confused with chronic follicular conjunctivitis (Table 1). The most frequently encountered conditions that are confused with chronic chlamydial conjunctivitis are now contact lens-induced giant papillary conjunctivitis, other forms of atopic conjunctivitis such as vernal catarrh and atopic keratoconjunctivitis, "toxic" follicular conjunctivitis caused by Molluscum contagiosum, and medications. As Dr. Sheppard rightly points out, topical therapy with an appropriate antibiotic does interfere with the laboratory diagnosis of chlamydial conjunctivitis, even though the clinical response may not be very substantial. Thus, it is important to know if the patient has been on previous antibiotic treatment with erythromycin, tetracycline, or a sulfonamide. Although Dr. Wiley and associates suggest that the adenovirus test might be a useful office procedure, the expense of

SHEPPARD et al

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ADULT CHLAMYDIAL CONJUNCTIVITIS

Table 1. Differential Diagnosis of Chronic Follicular Conjunctivitis Trachoma, inclusion conjunctivitis and other chlamydial infections "Toxic" follicular conjunctivitis Molluscum contagiosum Topical medications (idoxuridine, eserine, DFP, atropine, epinephrine, etc.) Eye make-up Moraxella and other bacteria Axenfeld's chronic follicular conjunctivitis Thygeson's chronic follicular conjunctivitis Parinaud's oculo-glandular fever Atopic keratoconjunctivitis Vernal catarrh Giant papillary conjunctivitis Rosacea keratoconjunctivitis

training office personnel and the use of an entire test kit with positive and negative controls for the individual case make the test inherently expensive. Thus, even this relatively simple test is best done by a clinical diagnostic laboratory that is running an adequate number of specimens to provide a sufficient quality control of the procedure. This is equally true of the currently available tests for C. trachomatis infection, all of which demand highly skilled laboratory personnel, even for the direct observation of Giemsa-stained conjunctival smears. There is no question that a simple test for use in the office would aid ophthalmologists substantially in the management of their cases. Such tests would have to be simplified so that they would be like the mononucleosis spot test or a dipstick test for glucose. Both groups have done excellent clinical research in identifying and examining the current means for diagnosing various forms of nonbacterial conjunctivitis in the laboratory.

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