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Comparative Evaluation of Methods for Laboratory Diagnosis of Herpetic Eye Disease
COMPARATIVE EVALUATION O F M E T H O D S FOR LABORATORY DIAGNOSIS O F H E R P E T I C EYE DISEASE N. S. ZAITSEVA, DR. SCI. ( M E D . ) , T. V. MURAVIEVA, C. SCI. ( B I O L . ) , V. L. VINOGRADOVA, C. Sci. ( M E D . ) , A. K. SHUBLADZE, DR. SCI. ( M E D . ) , T. M. MAEVSKAYA, C. Sci. ( M E D . ) , A. A. KASPAROV, C. SCI. ( M E D . ) , AND O. V. RZHECHITSKAYA, C. SCI. ( M E D . )
Moscow, U.S.S.R. Biomicroscopic examination is the most widely used method of diagnosis of ocular herpes simplex. However, there are many reports of other laboratory methods, includ ing virus isolation,1"5 cytologie examinations of conjunctival scrapings,2'6"9 fluorescent antibody ( F A ) techniques,10-15 intradermal tests with herpetic antigen,16"18 assessment of ocular reaction after intradermal injection of the antigen,19 as well as sérologie tests.18·20-21 This paper reports our evaluation of the significance of various laboratory methods currently used to diagnose and to investigate various aspects of the pathogenesis of her petic eye disease. MATERIALS AND METHODS
The fluorescent antibody technique and cytologie examinations of the conjunctival scrapings were used in examining 438 pa tients. Conjunctival scrapings were collected with a blunt scalpel on two slides. After fixation with methyl alcohol, one preparation was stained with Giemsa-Romanovsky stain. After fixation with cold acetone, the other was stained with rabbit immune serum labeled with fluorescein isothiocyanate (stain ing titer 1:8). Virus isolation was performed by intracerebral inoculation of white mice and inocu lation of tissue cultures with conjunctival scrapings from 26 patients. Immunologie tests—Intradermal tests with herpetic polyantigen were made in 255 From the Helmholtz Research Institute of Eye Disease, Moscow, U.S.S.R. Reprint requests to N. S. Zaitseva, M.D., Helm holtz Research Institute, Moscow, U.S.S.R.
patients. The polyantigen was prepared from the chorioallantoic membranes of chick em bryos infected with herpes virus strains of six antigenic groups and inactivated with 1 : 2000 formalin for seven days. Patients were inoculated with 0.05 ml of the polyantigen and the control antigen intradermally in their left and right arms, respectively. Tests were read 24 hours later when the skin reaction was maximal. The test was considered posi tive, if the maximum diameter of the hyperemic zone was at least 5 mm larger at the site of the polyantigen inoculation than that of the control antigen. Then the average values and the significance of their differences were determined. A focal allergic test was made in 61 pa tients suspected of having herpetic keratitis, keratoiridocyclitis, and iridocyclitis. The poyantigen (0.05 ml) was inoculated intra dermally twice, four days apart. The inten sity of the intradermal reaction was regis tered, and in order to detect any focal reac tions, the eyes of these patients were exam ined biomicroscopically each day. Serologie tests—Using serum samples from 74 patients, neutralization tests were performed in chick embryo fibroblast tissue cultures with the I-C strain of herpes virus.* The complement fixation test was performed in 195 patients with tissue culture antigens of herpes virus fixed for 18 hours at +4°C. RESULTS
The herpes antigen in the conjunctiva (Fig. 1) was detected in herpetic keratitis in 55.6% of patients in superficial forms in * Supplied by the Moscow Region Research Insti tute of Clinical Investigations. 997
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AMERICAN JOURNAL OF OPHTHALMOLOGY
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tologic examination of conjunctival scrap ings consisted of a lymphoid reaction with the presence of moderate and large lympho cytes (Fig. 3-A), plasma cells, and monocytes (Fig. 3-B). Alteration in the struc ture of lymphoid cells with increased basophilia of the cytoplasm and the presence of extracellular detritis were regularly ob served. Lymphoid reactions were observed more frequently (52.3%) in scrapings from the patients in whose conjunctiva the herpes antigen was found than those in whom no antigen was found (21.2%). These differ ences were not significant, however, and in scrapings from a considerable number of the patients a noncharacteristic cytologie picture was observed, with only a small number of neutrophils and lymphocytes detected. In the absence of herpes antigen this uncharacteris tic cytology was found in 42%, and when the antigen was detected it was observed in 26.4%. "Ballooning" degeneration of the epi thelium (Fig. 3-C) was observed irregularly and was found in the presence of the virus in the conjunctiva in 27.9%, and in the ab sence of the virus only in 3.5%. Signs of Fig. 1 (Zaitseva and associates). Fluorescence of virus antigen in the epithelium of conjunctival scrapping from a patient with herpetic keratitis. Direct FA technique. Top: Cytoplasm. Bottom: Nucleus (X210).
62.4%, and in profound forms in 44.3%. The antigen was found in the conjunctiva of 24.3% of the patients suspected of having herpetic infection of the eyes, and in the ker atitis of unknown etiology antigen was found in 7.8% (Table 1). It was detected in pa tients with conjunctivitis and in the corneal tissue removed in keratoplasty operations. Utilization of the FA technique facilitated the diagnosis in those cases in which clinical diagnosis was uncertain. In this study, we also analyzed the associa tion of cytologie reactions with the presence of herpes antigen in the conjunctival scrappings (Fig. 2 ) . The signs most frequently observed in cy-
TABLE 1 DETECTION OF HERPES ANTIGEN BY THE FLUORESCENT ANTIBODY TECHNIQUE IN DIFFERENT FORMS OF KERATIT1S, CONJUNCTIVITIS, AND IN CONTROL GROUPS
Clinical Diagnosis Herpetic keratitis: Superficial forms Profound forms Suspected herpetic keratitis: Superficial forms Profound forms Keratitis of unknown etiology Cataracts before keratoplasty Conjunctivitis Iridocyclites Control group: Patients with cataracts, glaucoma Normal subjects Total
No.
Antigen Detected
125 70
78(62.4%) 31(44.3%)
31 47 64 17 9 7
8(25.8%) 11(23.4%) S( 7.8%) 3 — 1 — 0
Patie°nts
30 30 438
0 0 158
999
HERPES SIMPLEX
VOL. 75, NO. 6
Herpetic superficial keratitis
60
52.3
50
42.0
40 26.4
30 20 7.0
10 J
0
L
c
60
8 '5i _o o l·
50.2 50 40 30
I
20 10 0 60 50
Suspect for herpes, profound keratitis
40 28.8
30 21.3 20
MIL3 8.7
10 0
8.7 '
0 0 ■■
1
Fig. 2 (Zaitseva and associates). Comparison of the frequency of detection of main cytological signs in the presence or absence of herpes antigen in the conjunctiva. (1) degeneration of epithelium, (2) multinuclear cells, (3) lymphoid reaction, (4) plasma cell reaction, (S) leukocytic reaction, (6) noncharacteristic reaction. Key: ■ = presence of antigen in the conjunctiva, O = absence of antigen in the conjunctiva.
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Fig. 3 (Zaitseva and associates). Cytologie picture of the conjunctival scrapping from a patient with herpetic keratitis. (A) lymphoid reaction, (B) plasma cell reaction, (C) degeneration of nuclei of the epithelial cells, and (D) multinuclear cell (Giemsa-Romanovsky stain, χξ>00).
dystrophy of the epithelium were found in 17.3% of patients with keratitis of unknown etiology. Dystrophie changes of the epithelium can also be found in allergic conjunctivitis which reduces the diagnostic value of this sign. Multinuclear cells (Fig. 3-D) were found predominantly in deep keratitis (29.9%) and much less frequently in superficial keratitis (6.9-9.8%). The cellular reaction with pre dominance of neutrophils was found in 17.8% of the patients with deep keratitis, and in 7% with superficial keratitis. The presence of eosinophils and basophils was observed in 1.6-3.5% of the patients. Comparing the cytologie picture in her petic infection of the eyes with that of other diseases of the conjunctiva and cornea, we were impressed that cytology is valuable in differentiating viral, allergic, and bacterial diseases. But based on our observations, weconcluded that the diagnosis of herpetic eye
disease can only be established specifically by detection of herpes antigen in conjuncti val scrapings or isolation of the causative agent. Comparison of the results of herpes virus detection by the FA technique and by means of virus isolation by the method we described, demonstrated the higher sensitivity of the FA technique. By virus isolation, herpes was found in 10 out of 26 patients, whereas her pes antigen was found in conjunctival scrap ings in 13 patients in this same group. Comparison and analysis of the diagnostic value of immunologie and sérologie methods shows that the rate of positive intradermal tests is slightly higher in deep keratitis (34.2-39.3%) than in superficial forms (23.2-33.3%) (Table 2 ) , with positive reac tions insignificantly higher in patients with recurrence. The intensity of reaction ex pressed in average arithmetic values and their confidence limits ranged from 6.85 ±
VOL. 75, NO. 6
HERPES SIMPLEX
1001
TABLE 2 RESULTS OF IMMUNOLOGIC STUDIES
Intradermal Test Groups Examined
Subjects
%
Positive: Superficial form: First attack 58 Relapse 66 Profound forms: 38 First attack Relapse 33 Control group: 60 With extraocular manifestations of herpes 28 Without clinical manifestations of herpes 32 Total 255
Polyantigen
Control Antigen
% Antigen Detected in Conjunctiva
Mean Titers in CFT
23.2 33.3
6.85 + 1.73 5.11 + 1.5 8.15 + 2.36 4.61±1.14
39.5 54.5
1:14.8 1:13.0
34.2 39.3
9.55+3.69 6.91+2.45 8.8 ± 2 . 6
5.58±2.5 3.55±1.32 4.8 ±1.9
30.0 43.5
1:19.2 1:20.8
43
10.3 ±3.71 6.3 ±3.37
—
1:19.5
28
7.4 ±3.75 3.5 ±2.73
—
1:8.8
35
—-
—
1.73 to 9.55 ± 3.69; differences in the inten of virus antigen in the conjunctiva and the sity of reaction to the specific and control results of intradermal testing (140 patients antigens are significant with a probability of examined) shows that the most frequent 99% and 95%. combination found in negative skin tests and Comparison of the rate and extent of posi negative attempt at antigen detection tive reactions to the specific allergen in (62.9%). Positive detection of virus antigen patients with herpetic eye disease, in patients and negative intradermal tests were found in with extraocular manifestations of herpes, 16.8% of the subjects examined. Positive in and in subjects without a history of herpetic tradermal tests were observed both in the infection revealed no significant differences. presence of antigen in the conjunctiva The values of intradermal reaction were (13.3%) and in its absence (9.3%). These slightly higher in patients with extraocular results indicate the lack of direct association manifestations of herpetic infection, how between detection of virus antigen in the ever. Individual variations in the intensity of conjunctiva and the results of the intrader reaction were observed in some patients, mal test. particularly patients within deep keratitis Focal herpetic polyantigen—Among 61 during the first attack. patients with suspected herpes infection of No definite correlation could be found be the eye, a clear-cut reaction developed in 17 tween the intensity of the intradermal test, (27.8%) (Table 3 ) . In the majority of them frequency of detection of virus antigen in it was manifested by increased intensity of the conjunctiva, and titers of complement- the corneal syndrome, pericorneal injection, fixing (CF) antibody. Intradermal reactions appearance of fresh corneal infiltrates, as and titers of CF antibody reach high levels well as precipitates and appositions on the in deep forms of the disease ; on the average, posterior surface of the cornea. Focal reac however, CF antibody titers were not high tion was observed not only in patients with (13.4:1:20.5). severe corneal and uveal tract infection, but Titers of virus-neutralizing antibody were also in those with iridocyclitis without the constantly high both in herpes patients and corneal involvement. Focal reaction was suc in the control group of patients with paratra- cessfully interrupted by subsequent, antiviral and desensitizing therapy in all 17 of these choma. Analysis of correlations between detection patients.
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AMERICAN JOURNAL OF OPHTHALMOLOGY
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TABLE 3 RESULTS OF SYNDROMIC REACTION AFTER INTRADERMAL INOCULATION OF ANTIGEN
AND
IMMUNOFLUORESCENT STUDIES IN PATIENTS S U S P E C T ' F O R HERPETIC EYE DISEASE
Clinical Diagnosis
No. Patients
Keratoconjunctivitis, keratitis 12 Keratoiridocyclitis 27 Episcleritis 2 Isolated iridocyclitis 16 Chorioiditis 4 Total 61 (100%)
Positive Results Syndromic Reaction 2 10 0 5 0 17 (27.8%)
FA 4 11 1 1 0 17 (27.8%)
Significance of Differences Chi2 = 0.9 0.5
Reactivation of infection in the eye in re In the present investigation, besides evalu sponse to intradermal inoculation of herpes ating the diagnostic significance of various antigen confirms the presence of latent in laboratory methods, we also obtained some fection capable of exacerbation upon extra data on the pathogenesis of the herpetic eye ocular antigenic stimulation. Our data provide evidence that the patho disease. genesis of herpetic eye disease as well as re Detection of the lymphoid-plasma cell re currences are determined by the effect of action in the conjunctiva, as well as positive multiple factors. Most important among intradermal and focal reactions in the eye in these are virus persistence in the tissues and response to administration of herpes antigen the mechanism of specific hypersensitivity. in patients with keratitis and iridocyclitis, The influence of provoking factors of both suggest a mechanism of hypersensitivity of specific and nonspecific nature is of great the delayed type in the pathogenesis of this 20 22 ' including the effect|of dif importance, disease. ferent kinds of stress on development recur At the same time, our investigation dem onstrated that reactions of immune type are rence (cooling, overheating, hormonal and not directly associated with the presence of nervous disorders). Detection of herpes antigen in conjunctithe virus in the conjunctiva or with skin hy val scrapings by the fluorescent antibody persensitivity to herpes antigen. Focal reactions, in the circumstances un technique is the most effective method of der which we observed them, provided an in laboratory diagnosis of herpetic keratitis and direct indication that exacerbation of the conjunctivitis. This technique is rapid, tech process in the eye after extraocular antigen nically simple, and suitable for use in the may be due both to reactivation of the infec clinic. Examination of conjunctival scrapings tion and to the development of cellular hy provides an additional diagnostic method, persensitivity. establishing features of cellular reaction as The lack of the direct association between well as distinguishing eye diseases of viral, virus detection in the conjunctiva and inten bacterial, and allergic nature. Of diagnostic sity of intradermal reactions, and enhance significance in herpes are: polymorphonument of the intensity of the latter in extraoc ular herpes, suggest that these reactions clear vacuolization of epithelial nuclei, detec characterize the immune response of the or tion of intranuclear inclusion bodies, multiganism to the presence of the virus not only nuclear cells, lymphoid-plasma cell reaction in in the eye but in extraocular tissues as well. conjunctiva tissue. DISCUSSION
HERPES SIMPLEX
VOL. 75, NO. 6
Intradermal tests with herpes polyantigen as well as sérologie tests (complement fixa tion and neutralization) have no diagnostic importance and should be reserved for use in the study of the pathogenesis of herpetic dis ease. SUMMARY
I n evaluating various laboratory methods for diagnosis of herpetic eye disease, it was established that the fluorescent antibody technique is the most effective. I n examina tions of 438 patients, herpes antigen was de tected in superficial keratitis in 62.4%, in deep forms in 4 4 . 3 % , in cases of suspected herpetic keratitis in 2 3 . 4 % . Positive reaction of the eye after intradermal inoculation of polyantigen was observed in 17 out of 61 pa tients suspected of having herpetic eye dis ease. Direct comparison of the results of virus isolation and antigen detection by the fluor escent antibody technique indicated that the latter method had higher sensitivity. Cytologie examination was of importance as an auxiliary method, having diagnostic value in uncertain cases. Observation sug gestive of herpes included degeneration of nuclei of the epithelium, detection of intra nuclear inclusions, multinuclear cells, and lymphoid-plasma cell reaction in conjunctival tissue. T h e latter was the most common cellular reaction in the presence of herpes. Intradermal tests for herpes polyantigen, as well as complement-fixation and neutraliza tion tests, have no diagnostic significance and should be reserved for use in the study of the pathogenesis of the disease. REFERENCES
1. Voinov, I. N. : Detection of herpes virus in ex perimental uveakeratitis. Materials of Scient. Conf. Chelyabinsk Med. Inst. Chelyabinsk, April, 1964, p. 276. 2. Kaznelson, A. B. : Herpetic eye diseases. Medizina (Leningrad), 1:104, 1969. 3. Maevskava, T. M., and Lavrentieva, A. M.: Isolation of herpes virus from patients with herpetic keratitis. Vopr. Virusol. 2:216, 1964. 4. White, D. O., Shew, M. A., Howsam, K. G.,
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and Robertson, J. J. : Herpes simplex virus infec tion of the cornea. Med. J. Aust. 22 :S9, 1968. 5. Coleman, V. R., Thygeson, P., Dawson, C. R., and Jawetz, E. : Isolation of virus from herpetic keratitis. Arch. Ophth. 81:22, 1969. 6. Kimura, S. J., and Thygeson, P. : The cytol ogy of external ocular disease. Am. J. Ophth. 39: 137, 19SS. 7. Thygeson, P . : Cytologie observations on her petic keratitis. Am. J. Ophth. 45:240, 1958. 8. Tarasova, A. N. : Cytological changes in the epithelium of the cornea and the conjunctiva in her petic eye disease. Vestn. Ophth. 1:49, 1966. 9. Voronova, N. S., and Glukhovskaya, S. S. : On the possibility of virus carries state in the conjunctival epithelium in herpes of the eye. Vestn. Ophth. 6:82,1969. 10. Kaufman, H. E. : Diagnosis of corneal herpes simplex infection by fluorescent antibody staining. Arch. Ophth. 64:382, 1960. 11. Pettit, T. H., Kimura, S. J., and Peters, H. : The fluorescent antibody technique in diagnosis of herpes simplex keratitis. Arch. Ophth. 72:86, 1964. 12. Benda, R., Hronovsky, V., Myska, V., Prochazka, O., Cerva, L. O., and Dubanska, H.: Ex periences with the method of fluorescent antibodies in the diagnosis of human herpetic keratoconjunctivitis. Ceskoslovenska. Cs. Epidem. 14:257, 1965. 13. Myska, V., and Benda, R. : Diagnostika herpetichych onemocheni rohovky technikou fluoreskujicich rotilatek. Cs. Optai. 22:2, 1966. 14. Muravieva, T. V., Maevskaya, T. M., Gushchina, L. V., Chibisova, V. A., and Krichevskaya, G. I. : Utilization of the fluorescent antibody techniqne for diagnosis of herpetic kératites and conjunctivites. Materials of the 28th Jubilee Conference of Helmholtz Institute of Eye Diseases, Moscow, 1967. 15. Krichevskaya, G. I., and Kasparov, A. A. : Immunofluorescent and cytological examination of conjunctival scrappings from patients with herpetic eye disease. Vestn. Ophth. 1:36, 1970. 16. Nagler, F. P. O. : A specific cutaneous reac tion in persons infected with the virus of herpes simplex. J. Immunol. 48:213, 1944. 17. Panschereuski, D., and Rohde, B. : Zur Serol ogie und Therapie des Herpes simplex recidivans. Hautzart. 63:275, 1963. 18. Lavrentieva, A. M., and Maevskaya, T. M. : The importance of skin tests in the diagnosis and treatment of herpetic keratitis. Vestn. Ophth. 1:45, 1966. 19. Kasparov, A. A. : Focal reaction to herpetic polyantigen as a new method for diagnosis of her petic eye infection. Collected methodical letters of first Moscow Medical Institute, Moscow, 1970. p. 83. 20. Leopold, I. H., and Sery, T. W. : Epidemiol ogy of herpes simplex keratitis. Invest. Ophth. 2: 498, 1963. 21. Smith, I. W., Peutherer, J. F., and MacCallum, F. O. : The incidence of herpesvirus hominis antibody in the population. J. Hyg. 65:395, 1967. 22. Laibson, P. R., and Kibrick, S. : Reactivation of herpetic keratitis in rabbit. Arch. Ophth. 77:244, 1967.
Moscow, U.S.S.R. Biomicroscopic examination is the most widely used method of diagnosis of ocular herpes simplex. However, there are many reports of other laboratory methods, includ ing virus isolation,1"5 cytologie examinations of conjunctival scrapings,2'6"9 fluorescent antibody ( F A ) techniques,10-15 intradermal tests with herpetic antigen,16"18 assessment of ocular reaction after intradermal injection of the antigen,19 as well as sérologie tests.18·20-21 This paper reports our evaluation of the significance of various laboratory methods currently used to diagnose and to investigate various aspects of the pathogenesis of her petic eye disease. MATERIALS AND METHODS
The fluorescent antibody technique and cytologie examinations of the conjunctival scrapings were used in examining 438 pa tients. Conjunctival scrapings were collected with a blunt scalpel on two slides. After fixation with methyl alcohol, one preparation was stained with Giemsa-Romanovsky stain. After fixation with cold acetone, the other was stained with rabbit immune serum labeled with fluorescein isothiocyanate (stain ing titer 1:8). Virus isolation was performed by intracerebral inoculation of white mice and inocu lation of tissue cultures with conjunctival scrapings from 26 patients. Immunologie tests—Intradermal tests with herpetic polyantigen were made in 255 From the Helmholtz Research Institute of Eye Disease, Moscow, U.S.S.R. Reprint requests to N. S. Zaitseva, M.D., Helm holtz Research Institute, Moscow, U.S.S.R.
patients. The polyantigen was prepared from the chorioallantoic membranes of chick em bryos infected with herpes virus strains of six antigenic groups and inactivated with 1 : 2000 formalin for seven days. Patients were inoculated with 0.05 ml of the polyantigen and the control antigen intradermally in their left and right arms, respectively. Tests were read 24 hours later when the skin reaction was maximal. The test was considered posi tive, if the maximum diameter of the hyperemic zone was at least 5 mm larger at the site of the polyantigen inoculation than that of the control antigen. Then the average values and the significance of their differences were determined. A focal allergic test was made in 61 pa tients suspected of having herpetic keratitis, keratoiridocyclitis, and iridocyclitis. The poyantigen (0.05 ml) was inoculated intra dermally twice, four days apart. The inten sity of the intradermal reaction was regis tered, and in order to detect any focal reac tions, the eyes of these patients were exam ined biomicroscopically each day. Serologie tests—Using serum samples from 74 patients, neutralization tests were performed in chick embryo fibroblast tissue cultures with the I-C strain of herpes virus.* The complement fixation test was performed in 195 patients with tissue culture antigens of herpes virus fixed for 18 hours at +4°C. RESULTS
The herpes antigen in the conjunctiva (Fig. 1) was detected in herpetic keratitis in 55.6% of patients in superficial forms in * Supplied by the Moscow Region Research Insti tute of Clinical Investigations. 997
998
AMERICAN JOURNAL OF OPHTHALMOLOGY
JUNE, 1973
tologic examination of conjunctival scrap ings consisted of a lymphoid reaction with the presence of moderate and large lympho cytes (Fig. 3-A), plasma cells, and monocytes (Fig. 3-B). Alteration in the struc ture of lymphoid cells with increased basophilia of the cytoplasm and the presence of extracellular detritis were regularly ob served. Lymphoid reactions were observed more frequently (52.3%) in scrapings from the patients in whose conjunctiva the herpes antigen was found than those in whom no antigen was found (21.2%). These differ ences were not significant, however, and in scrapings from a considerable number of the patients a noncharacteristic cytologie picture was observed, with only a small number of neutrophils and lymphocytes detected. In the absence of herpes antigen this uncharacteris tic cytology was found in 42%, and when the antigen was detected it was observed in 26.4%. "Ballooning" degeneration of the epi thelium (Fig. 3-C) was observed irregularly and was found in the presence of the virus in the conjunctiva in 27.9%, and in the ab sence of the virus only in 3.5%. Signs of Fig. 1 (Zaitseva and associates). Fluorescence of virus antigen in the epithelium of conjunctival scrapping from a patient with herpetic keratitis. Direct FA technique. Top: Cytoplasm. Bottom: Nucleus (X210).
62.4%, and in profound forms in 44.3%. The antigen was found in the conjunctiva of 24.3% of the patients suspected of having herpetic infection of the eyes, and in the ker atitis of unknown etiology antigen was found in 7.8% (Table 1). It was detected in pa tients with conjunctivitis and in the corneal tissue removed in keratoplasty operations. Utilization of the FA technique facilitated the diagnosis in those cases in which clinical diagnosis was uncertain. In this study, we also analyzed the associa tion of cytologie reactions with the presence of herpes antigen in the conjunctival scrappings (Fig. 2 ) . The signs most frequently observed in cy-
TABLE 1 DETECTION OF HERPES ANTIGEN BY THE FLUORESCENT ANTIBODY TECHNIQUE IN DIFFERENT FORMS OF KERATIT1S, CONJUNCTIVITIS, AND IN CONTROL GROUPS
Clinical Diagnosis Herpetic keratitis: Superficial forms Profound forms Suspected herpetic keratitis: Superficial forms Profound forms Keratitis of unknown etiology Cataracts before keratoplasty Conjunctivitis Iridocyclites Control group: Patients with cataracts, glaucoma Normal subjects Total
No.
Antigen Detected
125 70
78(62.4%) 31(44.3%)
31 47 64 17 9 7
8(25.8%) 11(23.4%) S( 7.8%) 3 — 1 — 0
Patie°nts
30 30 438
0 0 158
999
HERPES SIMPLEX
VOL. 75, NO. 6
Herpetic superficial keratitis
60
52.3
50
42.0
40 26.4
30 20 7.0
10 J
0
L
c
60
8 '5i _o o l·
50.2 50 40 30
I
20 10 0 60 50
Suspect for herpes, profound keratitis
40 28.8
30 21.3 20
MIL3 8.7
10 0
8.7 '
0 0 ■■
1
Fig. 2 (Zaitseva and associates). Comparison of the frequency of detection of main cytological signs in the presence or absence of herpes antigen in the conjunctiva. (1) degeneration of epithelium, (2) multinuclear cells, (3) lymphoid reaction, (4) plasma cell reaction, (S) leukocytic reaction, (6) noncharacteristic reaction. Key: ■ = presence of antigen in the conjunctiva, O = absence of antigen in the conjunctiva.
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AMERICAN JOURNAL OF OPHTHALMOLOGY
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Fig. 3 (Zaitseva and associates). Cytologie picture of the conjunctival scrapping from a patient with herpetic keratitis. (A) lymphoid reaction, (B) plasma cell reaction, (C) degeneration of nuclei of the epithelial cells, and (D) multinuclear cell (Giemsa-Romanovsky stain, χξ>00).
dystrophy of the epithelium were found in 17.3% of patients with keratitis of unknown etiology. Dystrophie changes of the epithelium can also be found in allergic conjunctivitis which reduces the diagnostic value of this sign. Multinuclear cells (Fig. 3-D) were found predominantly in deep keratitis (29.9%) and much less frequently in superficial keratitis (6.9-9.8%). The cellular reaction with pre dominance of neutrophils was found in 17.8% of the patients with deep keratitis, and in 7% with superficial keratitis. The presence of eosinophils and basophils was observed in 1.6-3.5% of the patients. Comparing the cytologie picture in her petic infection of the eyes with that of other diseases of the conjunctiva and cornea, we were impressed that cytology is valuable in differentiating viral, allergic, and bacterial diseases. But based on our observations, weconcluded that the diagnosis of herpetic eye
disease can only be established specifically by detection of herpes antigen in conjuncti val scrapings or isolation of the causative agent. Comparison of the results of herpes virus detection by the FA technique and by means of virus isolation by the method we described, demonstrated the higher sensitivity of the FA technique. By virus isolation, herpes was found in 10 out of 26 patients, whereas her pes antigen was found in conjunctival scrap ings in 13 patients in this same group. Comparison and analysis of the diagnostic value of immunologie and sérologie methods shows that the rate of positive intradermal tests is slightly higher in deep keratitis (34.2-39.3%) than in superficial forms (23.2-33.3%) (Table 2 ) , with positive reac tions insignificantly higher in patients with recurrence. The intensity of reaction ex pressed in average arithmetic values and their confidence limits ranged from 6.85 ±
VOL. 75, NO. 6
HERPES SIMPLEX
1001
TABLE 2 RESULTS OF IMMUNOLOGIC STUDIES
Intradermal Test Groups Examined
Subjects
%
Positive: Superficial form: First attack 58 Relapse 66 Profound forms: 38 First attack Relapse 33 Control group: 60 With extraocular manifestations of herpes 28 Without clinical manifestations of herpes 32 Total 255
Polyantigen
Control Antigen
% Antigen Detected in Conjunctiva
Mean Titers in CFT
23.2 33.3
6.85 + 1.73 5.11 + 1.5 8.15 + 2.36 4.61±1.14
39.5 54.5
1:14.8 1:13.0
34.2 39.3
9.55+3.69 6.91+2.45 8.8 ± 2 . 6
5.58±2.5 3.55±1.32 4.8 ±1.9
30.0 43.5
1:19.2 1:20.8
43
10.3 ±3.71 6.3 ±3.37
—
1:19.5
28
7.4 ±3.75 3.5 ±2.73
—
1:8.8
35
—-
—
1.73 to 9.55 ± 3.69; differences in the inten of virus antigen in the conjunctiva and the sity of reaction to the specific and control results of intradermal testing (140 patients antigens are significant with a probability of examined) shows that the most frequent 99% and 95%. combination found in negative skin tests and Comparison of the rate and extent of posi negative attempt at antigen detection tive reactions to the specific allergen in (62.9%). Positive detection of virus antigen patients with herpetic eye disease, in patients and negative intradermal tests were found in with extraocular manifestations of herpes, 16.8% of the subjects examined. Positive in and in subjects without a history of herpetic tradermal tests were observed both in the infection revealed no significant differences. presence of antigen in the conjunctiva The values of intradermal reaction were (13.3%) and in its absence (9.3%). These slightly higher in patients with extraocular results indicate the lack of direct association manifestations of herpetic infection, how between detection of virus antigen in the ever. Individual variations in the intensity of conjunctiva and the results of the intrader reaction were observed in some patients, mal test. particularly patients within deep keratitis Focal herpetic polyantigen—Among 61 during the first attack. patients with suspected herpes infection of No definite correlation could be found be the eye, a clear-cut reaction developed in 17 tween the intensity of the intradermal test, (27.8%) (Table 3 ) . In the majority of them frequency of detection of virus antigen in it was manifested by increased intensity of the conjunctiva, and titers of complement- the corneal syndrome, pericorneal injection, fixing (CF) antibody. Intradermal reactions appearance of fresh corneal infiltrates, as and titers of CF antibody reach high levels well as precipitates and appositions on the in deep forms of the disease ; on the average, posterior surface of the cornea. Focal reac however, CF antibody titers were not high tion was observed not only in patients with (13.4:1:20.5). severe corneal and uveal tract infection, but Titers of virus-neutralizing antibody were also in those with iridocyclitis without the constantly high both in herpes patients and corneal involvement. Focal reaction was suc in the control group of patients with paratra- cessfully interrupted by subsequent, antiviral and desensitizing therapy in all 17 of these choma. Analysis of correlations between detection patients.
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AMERICAN JOURNAL OF OPHTHALMOLOGY
JUNE, 1973
TABLE 3 RESULTS OF SYNDROMIC REACTION AFTER INTRADERMAL INOCULATION OF ANTIGEN
AND
IMMUNOFLUORESCENT STUDIES IN PATIENTS S U S P E C T ' F O R HERPETIC EYE DISEASE
Clinical Diagnosis
No. Patients
Keratoconjunctivitis, keratitis 12 Keratoiridocyclitis 27 Episcleritis 2 Isolated iridocyclitis 16 Chorioiditis 4 Total 61 (100%)
Positive Results Syndromic Reaction 2 10 0 5 0 17 (27.8%)
FA 4 11 1 1 0 17 (27.8%)
Significance of Differences Chi2 = 0.9 0.5
Reactivation of infection in the eye in re In the present investigation, besides evalu sponse to intradermal inoculation of herpes ating the diagnostic significance of various antigen confirms the presence of latent in laboratory methods, we also obtained some fection capable of exacerbation upon extra data on the pathogenesis of the herpetic eye ocular antigenic stimulation. Our data provide evidence that the patho disease. genesis of herpetic eye disease as well as re Detection of the lymphoid-plasma cell re currences are determined by the effect of action in the conjunctiva, as well as positive multiple factors. Most important among intradermal and focal reactions in the eye in these are virus persistence in the tissues and response to administration of herpes antigen the mechanism of specific hypersensitivity. in patients with keratitis and iridocyclitis, The influence of provoking factors of both suggest a mechanism of hypersensitivity of specific and nonspecific nature is of great the delayed type in the pathogenesis of this 20 22 ' including the effect|of dif importance, disease. ferent kinds of stress on development recur At the same time, our investigation dem onstrated that reactions of immune type are rence (cooling, overheating, hormonal and not directly associated with the presence of nervous disorders). Detection of herpes antigen in conjunctithe virus in the conjunctiva or with skin hy val scrapings by the fluorescent antibody persensitivity to herpes antigen. Focal reactions, in the circumstances un technique is the most effective method of der which we observed them, provided an in laboratory diagnosis of herpetic keratitis and direct indication that exacerbation of the conjunctivitis. This technique is rapid, tech process in the eye after extraocular antigen nically simple, and suitable for use in the may be due both to reactivation of the infec clinic. Examination of conjunctival scrapings tion and to the development of cellular hy provides an additional diagnostic method, persensitivity. establishing features of cellular reaction as The lack of the direct association between well as distinguishing eye diseases of viral, virus detection in the conjunctiva and inten bacterial, and allergic nature. Of diagnostic sity of intradermal reactions, and enhance significance in herpes are: polymorphonument of the intensity of the latter in extraoc ular herpes, suggest that these reactions clear vacuolization of epithelial nuclei, detec characterize the immune response of the or tion of intranuclear inclusion bodies, multiganism to the presence of the virus not only nuclear cells, lymphoid-plasma cell reaction in in the eye but in extraocular tissues as well. conjunctiva tissue. DISCUSSION
HERPES SIMPLEX
VOL. 75, NO. 6
Intradermal tests with herpes polyantigen as well as sérologie tests (complement fixa tion and neutralization) have no diagnostic importance and should be reserved for use in the study of the pathogenesis of herpetic dis ease. SUMMARY
I n evaluating various laboratory methods for diagnosis of herpetic eye disease, it was established that the fluorescent antibody technique is the most effective. I n examina tions of 438 patients, herpes antigen was de tected in superficial keratitis in 62.4%, in deep forms in 4 4 . 3 % , in cases of suspected herpetic keratitis in 2 3 . 4 % . Positive reaction of the eye after intradermal inoculation of polyantigen was observed in 17 out of 61 pa tients suspected of having herpetic eye dis ease. Direct comparison of the results of virus isolation and antigen detection by the fluor escent antibody technique indicated that the latter method had higher sensitivity. Cytologie examination was of importance as an auxiliary method, having diagnostic value in uncertain cases. Observation sug gestive of herpes included degeneration of nuclei of the epithelium, detection of intra nuclear inclusions, multinuclear cells, and lymphoid-plasma cell reaction in conjunctival tissue. T h e latter was the most common cellular reaction in the presence of herpes. Intradermal tests for herpes polyantigen, as well as complement-fixation and neutraliza tion tests, have no diagnostic significance and should be reserved for use in the study of the pathogenesis of the disease. REFERENCES
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