ACTA O P H T H A L M O L O G I C A VOL. 55 1977

Departments of Ophthalmology (E. Palm) and Medical Microbiology (L. Kjellkn), University Hospital, Lund, Sweden



Fifty-seven patients with ocular herpes simplex (HS) infection were studied for evaluation of existing methods for virus isolation and its application in diagnosis of HS infection. Virus was isolated in 90 o/o of 34 cases with keratitis dendritica, when specimens were taken within eight days of onset of symptoms. The same isolation frequency was obtained in 10 cases of palpebral herpes with conjunctivitis. No isolation was possible in 11 cases of keratitis disciformis. Laboratory confirmation was obtained within four days in 70°/o of the positive cases. Ten strains of HS virus type 1 were examined for IDU resistance, 5 strains isolated prior to and 5 during IDU treatment. Nine of the strains had the same degree of sensitivity. One strain isolated during treatment was found to be highly resistant.

Key words: ocular herpes simplex - virus isolation

- IDU resistance.

Treatment of ocular herpes simplex (HS) infection is still a problem. Kaufman introduced 5-iodo-2-deoxyuridine (IDU) in 1962 as an antiviral compound for these infections (Kaufman 1962). Several investigations have shown its clinical value (Kaufman 1965), and IDU is often a preferable treatment in combination with traditional treatment like abrasio corneae and cauterization. The clinically IDU resistant cases pose a special problem and the question arises as to whether IDU resistant strains of HS virus have developed. In vitro Received May 10, 1977.

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L. Nordenfelt and E. Nordenfelt

studies have shown that such strains can easily develop (Buthala 1964). Clinical reports have stated the existence of resistant strains in single cases (Laibson et al. 1963; Pavan-Langston et al. 1972). Because of these findings it has been assumed that IDU resistance is m e important cause of therapy failure. This, hcwever, is a moot point (Jawetz et al. 1970). Besides IDU, several other anti-viral compounds have been tested and are being introduced as alternatives in treatment of ocular HS infections (Kaufman 1976). The present investigation was made in order to study existing methods for virus isolation in diagnosis of ocular HS infection in view of the need for laboratory diagnosis in evaluating anti-viral drugs during testing. Furthermore, a study was done on IDU-resistance in HS strains isolated prior to and during IDU treatment.

Material The material consisted of 57 patients with the preliminary diagnosis of ocular HS infection. They were close to the total number of patients treated for this disease as out-patients as well as in-patients at the Eye Clinic, University Hospital of Lund, during an eighteen month period. The patients were divided into different groups according to the clinical picture. Thirtyfour had a keratitis with typical dendritical lesions of the epithelium, 10 patients had vesicular eruptions on the lids with conjunctivitis and 1 1 cases had keratitis disciformis with blurring of the stroma. Two cases had a keratitis with punctata lesions of unspecific type. Three patients had a recurrence of infection during the time of investigation. The recurrences were regarded as separate cases. Ten strains of isolated HS virus were examined for resistance to IDU. Five strains were isolated before start of therapy. Five were isolated during IDU treatment.

Methods In most cases the first specimen was obtained at the first examination after start of symptoms. Cauterization or abrasio corneae was not performed until after the specimens had been obtained. At the start of the investigation specimens were obtained both by scraping the cornea and by turning a cotton-tipped swab in the lower fornix. The isolation frequency was the same with both procedures. During the main part of the investigation the specimens were obtained with the swab, because of the ease and harmlessness of this technique. 920

Herpes Simfilex

- Virus Isolation and IDU Resistance

A trypton broth was used as a transport medium. The specimen tubes were stored in a refrigerator at +4"C, and were brought to the virus laboratory once every weekday. I8olatlon procedures

Before inoculation of the specimens penicillin and streptomycin were added. If immediate inoculation could not be carried out, the samples were frozen at -70°C. Cell cultures in roller tubes were prepared from GMK (green monkey kidney) cells (Flow laboratories, Irvine, Scotland), MAS (cell line established from human bone marrow) cells (KjellCn 1961), and human embryonic diploid lung fibroblasts (HEL) prepared in our laboratory. Inoculation of specimens was made in duplicate roller culture tubes which were incubated at 37OC and observed three times weekly for cytopathic effect (CPE). Cultures showing no CPE within 14 days of incubation were discarded as negative. No blind passages were made. Typing was done by neutralization tests in roller culture tubes using guinea pig antisera obtained from the National Bacteriological Laboratory, Stockholm, Sweden. Evaluation of the in vitro sensitlvity of isolated herpes simplex strains

Virus strains were taken from the first passage after isolation and stored at -60°C. 0.2 ml of a dilution of 10-2 was inoculated in eight Carrel1 culture bottles. Two days prior to inoculation the bottles were seeded with 106 HEL cells/ml (Medium MEM 100/0 foetal calf serum Flow lab., Irvine, Scotland). At the time of inoculation the medium was changed and two bottles each received MEM with 2 O/O foetal calf with IDU (Ferring, Malmo, Sweden) at concentrations of 75, 50, 25, and 0 pglml. After two days all bottles were frozen and virus concentration from the different bottles was determined by titration in roller tube cultures of HEL cells with five tubes in each dilution. The titration end point was read after three days and 500/0 tissue culture infective dose (TCIDm) was determined according to the Reed-Muench formula.

In 57 patients with the preliminary diagnosis of ocular HS infection, HS virus type 1 was isolated in 30 cases (Table I). A preliminary laboratory answer of these positive isolations was obtained within 2-3 days in 40% and within 4 days in 70 O/O, and the rest within a week. 92 1

L. Nordenfelt and E. Nordenfelt Table I . Final diagnosis and relation to virus isolation in 57 patients with preliminary diagnosis ocular herpes simplex infection.



of patients


Keratitis dendritica Palpebral herpes with conjunctivitis Keratitis disciformis Atypical keratitis


Positive isolations



10 11


9 0 0



Of the 57 patients 34 had a final diagnosis of keratitis dendritica, and virus was isolated in 21 cases. The frequency of positive isolation was influenced by the time when specimens were taken after the first symptoms and by the fact that in some cases treatment with IDU was started before specimens were obtained (Table 11). If specimens were taken within eight days of the first symptoms and no treatment had been provided, 900/0 of the cases were positive (18 of 20 cases).

Table 11. Result of isolation trials in 34 patients with keratitis dendritica with regard to time after first symptoms and eventual IDU treatment prior to isolation. . 1-8 days no IDU


1-8 days IDU treatment

> 8 days with or without treatment


Positive Negative ~~

18 2 20

3 6 9


Total 34


0 5 5

Herpes Simplex - Virus Isolation and IDU Resistance

There were 10 patients with the diagnosis of palpebral herpes with conjunctivitis. In nine cases virus was isolated. Clinical diagnosis was made on the basis of symptoms of conjunctivitis with vesicular eruptions on the lids. None had developed keratitis before isolation. Half of the cases developed corneal epithelial lesions a few days after isolation. There were 11 cases with the final diagnosis of keratitis disciformis. Clinical diagnosis was made from blurring of the stroma as well as oedema of the epithelium. There was a history of keratitis dendritica in all cases. No case with actual lesions typical of dendritica was referred to this group. In no case virus was isolated. The remaining two cases had the preliminary diagnosis of keratitis herpetica? There was a history of earlier incidents. Subjectively, the symptoms were discomfort, and objectively fluorescein staining punctata lesions of unspecific type. They healed in a couple of days. In nine cases with positive virus isolation repeated isolations were done. In five cases these were positive. Re-isolation was done in four of these cases within eight days. In one case virus was isolated 4 times four days apart during IDU treatment. This patient suffered from leukaemia at a late stage. Treatment with IDU was begun after the first isolation in all cases. Ten strains from 10 patients were studied for their in vitro sensitivity to IDU. Five strains were isolated from patients who had not been treated with


F i g . I, Titer of ten strains of herpes simplex virus isolated prior to and during IDU treatment grown in 0, 25, 50, and 75 pglml IDU. ----- strains isolated prior to IDU treament. strains isolated during IDU treatment.


L. Nordenfelt and E. Nordenfelt

IDU and five were isolations done from patients during 4-6 times treatment with IDU daily. These patients were treated for 2 , 4 , 5, 7 and 12 days. Nine of the strains had the same degree of sensitivity and showed a 90 O/o or more reduction of infectivity already at 25 ,pg/ml IDU (Fig. I ) , whether the isolation was done prior to or during IDU treatment. One strain isolated after seven days of IDU treatment was not inhibited even by 50 pglml, a pattern distinctly different from the others (Fig. 1). From this patient an earlier isolate was available showing the same high degree of resistance to IDU. This isolation was done before IDU treatment was started.

Discussion The investigation showed that existing methods for virus isolation are useful in diagnosis of ocular HS infections. In the majority of cases there was a positive laboratory confirmation in four days or earlier. Duration of symptoms and treatment with IDU affects the frequency of isolation. In patients without IDU treatment and with duration of symptoms less than eight days the frequency of isolation was 18 of 20 (90 0/0). This is in agreement with earlier findings by Coleman et al. (1969) who found the highest isolation frequency (62 O/O) in this group of patients. Generally, it was no problem to establish the diagnosis in this particular type of ocular HS infection, but there is an advantage in using a reliable and fast isolation procedure when evaluating new antiviral agents. In the cases with palpebral herpes with conjunctivitis there is a greater interest in aides to obtain the correct diagnosis. This type is common among children and there are problems in differentiating for instance against impetigo contagiosum. Virus was isolated in 9 cases out of 10, although there were no corneal lesions. Treatment with IDU was begun after positive isolation. Only one case progressed to a classic dendritical lesion. The group of keratitis disciformis is a type of ocular HS infection where the differential diagnosis presents a problem if there is no earlier incident of herpetic keratitis and the symptoms are unspecific. In agreement with other reports (Coleman et al. 1969) we were unable to isolate virus in this deep stromal type of keratitis. At present very few data are available as to the emergence of drug resistant virus in man (Oxford 1976). In vitro studies have shown that resistant HS virus could easily be obtained by serial passage of the virus in cell culture in the presence of IDU (Buthala 1964). In agreement with this, in vivo studies in rabbits showed that 3 out of 4 isolates from corneal epithelium of eyes previously treated with IDU were highly resistant to IDU in vitro (Underwood et a1 1965). 924

Herpes Simplex - Virus Isolation and IDU Resistance

Single strains of herpes simplex isolated from clinical cases have been reported resistant to IDU when tested in vitro (Laibson 1963; Pavan-Langston 1972). Because of these reports it has been assumed that IDU resistance is one important cause of therapy failure. True drug resistance in unresponsive herpes keratitis, however, has not often been studied. In one investigation 12 HS strains from “clinically-IDU-resistant” patients were examined for drug-susceptibility in cell culture (Jawetz et al. 1970). Ten out of the 12 isolates were equally sensitive to IDU and two isolates were 10 to 30 times more resistant. In our investigation 4 out of 5 strains isolated during IDU treatment were as sensitive to IDU as five strains isolated before treatment. The one resistant strain was highly resistant to IDU, but the same resistance was obtained in an earlier isolate from the same patient before IDU treatment was started. Our results indicate that IDU resistance in wivo does not appear very easily. The frequency of resistance seems to be the same today as reported earlier (Jawetz et al. 1970) in spite of frequent use of IDU. Therapy failure may be caused by other factors, such as impaired immunological capacity. In this investigation, this is shown by the leukaemia case. In spite of full IDU treatment, during 12 days in hospital, an IDU sensitive HS strain was isolated.

References Buthala D. A. (1964) Cell culture studies on antiviral agents: Action of cytosine arabinoside and some comparison with 5-iodo-2-deoxyuridine. Proc. SOC.exp. Biol. ( N . Y.)115, 69-77. Coleman V. R., Thygeson P., Dawson Ch. & Jawetz E. (1969) Isolation of virus from herpetic keratitis. Arch. O@hthal. (Chicago) 81, 22-24. Jawetz E., Coleman V. R., Dawson C. K. & Thygeson P. (1970) The dynamics of IUDR action in herpetic keratitis, and the emergence of IUDR resistance in viuo. Ann. N . Y . Acad. Ski. 173, 282-289. Kaufman bl. E., Nesburn A. B. & Maloney E. D. (1962) IDU therapy of herpes simplex. Arch. Ophthal. (Chicago) 67, 583-591. Kaufman M. E. (1965) Problems in virus chemotherapy. Progr. med. Virol. 7, 116-159. Kaufman M. E. (1976) Ocular antiviral therapy in perspective. /. infect. Dis. I33 (Suppl.), A 96-A 100. KjellCn L. (1961) A study of Adenovirus-host cell system by the plaque technique. Virology 14, 234-237. Laibson P. R., Sery T. W. & Leopold I. H. (1963) The treatment of herpetic keratitis with 5-iodo-2-deoxyuridine (IDU). Arch. Ophthal. (Chicago) 70, 52-58. Pavan-Langston D. & Dahlman C. H. (1972) A double-blind clinical study of adenine therapy of viral keratoconjunctivitis. Amer. /. Ophthal. 74, 81-88.


L. Nordenfelt and E. Nordenfelt Oxford J. S. (1976) Drug resistance and antiviral agents. /. Antimicrobiol. Chemother. 2, 223-224. Underwood G. E., Elliott G. A. & Buthala D. A. (1965) Herpes keratitis in rabbits: Pathogenesis and effect of antiviral nucleosides. Ann. N . Y . Acad. Sci. 130, 151-167.

Author’s address: L. Nordenfelt, M. D., Department of Ophthalmology, University Hospital, S-223 62 Lund, Sweden.


Ocular herpes simplex infection. A clinical evaluation of virus isolation and studies on iodo-deoxyuridine resistance.

ACTA O P H T H A L M O L O G I C A VOL. 55 1977 Departments of Ophthalmology (E. Palm) and Medical Microbiology (L. Kjellkn), University Hospital, Lu...
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