Indian Journal of Ophthalmology

: 1996  |  Volume : 44  |  Issue : 1  |  Page : 19--21

An epidemic of viral acute haemorrhagic conjunctivitis in Delhi in 1994

Gita Satpathy, Sujata Mohanty, Niranjan Nayak 
 Dept. of Microbiology, Dr. R.P. Centre for Ophthalmic Sciences, A.I.I.M.S, New Delhi 110 029, India

Correspondence Address:
Gita Satpathy
Dept. of Microbiology, Dr. R.P. Centre for Ophthalmic Sciences, A.I.I.M.S, New Delhi 110 029


An epidemic of acute haemorrhagic conjunctivitis affecting persons of all ages and both sexes occurred in Delhi and surrounding areas during the monsoon season of 1994. The symptoms lasted on an average for 4-5 days. In some of the patients corneal involvement was observed. Conjunctival swabs from the affected patients were processed for viral antigen detection, virus isolation and bacterial culture and sensitivity. Viral antigen was detected in 62% (31/50) of the smears tested by indirect immunofluorescence assay. In 22 (44%) of the specimens Coxackie A 24 (Cox A 24) virus antigen and in 9 (18%) of the specimens Entero Virus 70 (EV 70) antigen were detected. In confluent monolayers of Hep 2 cells cytopathic virus was isolated in 10 (30.30%) of the 33 specimens processed. The isolated viruses were identified as either Cox A 24 (7 isolates) or EV 70 (3 isolates) using indirect immunofluorescence assay. Super added bacterial infection was observed in 33% (89/270) of the cases, Staphylococcus albus being the predominant bacteria isolated.

How to cite this article:
Satpathy G, Mohanty S, Nayak N. An epidemic of viral acute haemorrhagic conjunctivitis in Delhi in 1994.Indian J Ophthalmol 1996;44:19-21

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Satpathy G, Mohanty S, Nayak N. An epidemic of viral acute haemorrhagic conjunctivitis in Delhi in 1994. Indian J Ophthalmol [serial online] 1996 [cited 2022 Jun 28 ];44:19-21
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Viral conjunctivitis epidemics are reported from all over the world frequently.[1] Prior to 1970, Adeno virus 8 was the major known cause of epidemic outbreaks of acute viral conjunctivitis and keratoconjunctivitis. After 1970, acute haemorrhagic conjunctivitis epidemics are reported to be caused by Entero viruses; either Entero Virus 70 (EV 70), Coxackie Virus A 24, their variants or in combinations.[2],[3]

An epidemic of acute haemorrhagic conjunctivitis with characteristic clinical features of short incubation period, explosive onset, prominent sub conjunctival haemorrhage, swept through Delhi and surrounding areas during July to September 1994. The laboratoty investigations of this epidemic are reported here.



A total number of 270 patients of acute haemorrhagic conjunctivitis attending the Out Patients Department and the Casualty of Dr R P Centre for Ophthalmic Sciences from July to September 1994 were included in the study.

The age range of the patients varied from 2 to 59 years and there was an equal sex distribution.

The patients complained of pain, irritation and grittiness in the eye. There was redness and watering of conjunctiva. In most of the patients there was bilateral involvement. In some patients there was pre auricular lymphnode enlargement. In some cases there was corneal involvement in the form of superficial punctate keratitis. The symptoms lasted on an average for 4-5 days.

 Collection of Specimens

From the 50 of the affected patients conjunctival specimens were collected with sterile cotton tipped swabs and transported to the laboratory in 1.5ml of 0.2M sucrose phosphate buffer (2SP) pH7.2 for viral investigations.The specimens from one of the affected eye was studied from each patient for the virological studies. A swab was collected from each of the 270 patients attending the out patients department, in plain sterile vial for bacterial culture and sensitivity.

 Virological Investigations

 Immunofluorescence Assay for Viral Antigen Detection

The specimens collected from the 50 patients in 2 SP (One specimen from each patient) were cytocentrifuged onto clean glass slides in a cytocentrifuge (Shandon) on the same day of collection. The resulting smears of conjunctival cells were fixed in cold acetone for 10 minutes and stored at -20C till tested. An indirect immunofluorescence assay was done on the smears from these 50 patients in triplicate Hyper immune rabbit sera against EV 70 (at 1:10) (obtained by our laboratory from Dr Kono, Japan, in 1981 and preserved at -70C) and Coxackie A 24 (at 1:20) (obtained from National Institute of Virology, Pune, India) and goat anti adenovirus immunoglobulins (at 1:20) (Biodesign International, ME) were used as the primary antibodies in the assay. Goat anti rabbit immunoglobulins tagged with FITC at 1:32 (DAKO, UK) and rabbit anti goat immunoglobulins tagged with FITC at 1:32 (DAKO, UK) were used as the secondary antibodies. After mounting, the smears were examined under a fluorescent microscope (Nikon).

Conjunctival swabs collected from 10 normal individuals and processed as above were used as controls.

 Isolation of the Virus in Tissue Culture

Confluent monolayer of Hep 2 cells maintained in Minimum Essential Medium(GIBCO BRL,Life Technologies, UK) with 5% foetal bovine serum (Flow Laboratories, UK) was used for this purpose. Specimens collected in 2 SP from 33 patients (Specimen from one eye from each patient) were inoculated in duplicate in two tubes of Hep2 cells and incubated at 35C with frequent changing of the medium for a minimum period of 2 weeks. The cells were examined daily under an inverted microscope for appearance of cytopathic effect(CPE). The isolates from the culture tubes showing CPE were passaged once in Hep2 cells for confirmation of the specific viral CPE and concentration of the virus. Uninoculated tubes of Hep 2 cells were included as controls.

 Identification of the Virus Isolates

The Virus isolates were identified using the indirect immunofluorescence assay. The infected cells from the culture tubes showing CPE were harvested and washed in phosphate buffer saline (PBS). The cells were suspended in PBS pH 7.2 and smears were made by cytospinning the suspension in a Shandon cytocentrifuge. Indirect immunofluorescence assay was performed on the smears using rabbit antiserum against Cox A 24 and EV 70 at dilutions of 1:20 and 1:10 respectively.

 Bacterial Culture and Sensitivity

Conjunctival swabs from all the patients (270) were cultured on blood agar medium and thioglycolate broth for bacterial culture. The bacteria isolated were identified and their anti microbial sensitivity was obtained using standard procedures.[4]


In the immunofluorescence assay for antigen detection in conjunctival swabs, viral antigen could be detected in 31 of the 50 smears tested(62%). In 22 smears there was positive reaction with Cox A 24 antiserum (70.96%) and in 9 smears there was positive reaction with EV 70 antiserum (29.03%) (Table). None of the specimens gave positive result with adenovirus antiserum. In the immunofluorescence assay with either antiserum, there was a uniform cytoplasmic fluorescence of the conjunctival epithelial cells [Figure:1]. No fluorescence was observed with conjunctival specimens from normal individuals.

In the virus isolation procedure, cytopathic effect in the form of marked cell rounding of the inoculated Hep 2 cells was observed within 4 days of inoculation. The CPE was observed in 10 of the 33 specimens inoculated (30.30%).

In the indirect immunofluorescence assay done with the virus infected Hep 2 cells, 7 viral isolates gave positive reaction with Cox A 24 antiserum [Figure:2] and 3 viral isolates gave positive reaction with EV 70 antiserum. The former 7 specimens were positive for Cox A 24 antigen and the latter 3 specimens were positive for EV 70 antigen in the immunofluorescence assay for antigen detection.

In bacterial culture and sensitivity testing, bacteria were isolated from 89 of the 270 (32.96%) patients. Staphylococcus albus was the predominant bacteria isolated, 55/89 (61.79%); Staphylococcus aureus 5(5.61%); Acinetobacter spp.4(4.49%); Micrococcus spp. 10(11.23%); Diphtheroids 10(11.23%) and alpha haemolytic streptococci were the other bacteria isolated.


The seasonal occurrence and clinical features noted in this epidemic were similar to previous epidemics occurring in Delhi.[5, 6] However after 1977[7], this was the only instance where 2 different viruses were simultaneously involved in the conjunctivitis epidemics.

In Hep 2 cells, the present virus isolation rate of 30.03% is comparable to our previous isolation rate of 28.9% in BGMK cells[8] and the reported isolation rate of 30% in primary human embryonic kidney cells.[9] As has been observed earlier[10] the direct antigen detection method using indirect immunofluorescence assay, proved to be an effective tool for early diagnosis of the viral conjunctivitis, detecting antigen in 62% of the cases.


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