Clinical and molecular investigation of Acute Haemorrhagic Conjunctivitis outbreak in North India

doi:10.21203/rs.3.rs-4814766/v1

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Clinical and molecular investigation of Acute Haemorrhagic Conjunctivitis outbreak in North India

https://doi.org/10.21203/rs.3.rs-4814766/v1

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Purpose

The study aims to investigate an outbreak of acute haemorrhagic conjunctivitis (AHC) reported in North India.

Methods

All patients presenting to the outpatient clinic department with signs and symptoms (symptoms onset within 36 hours) of AHC were enrolled in the study. The conjunctival swab samples were tested for the presence of enteroviruses and adenoviruses. Detailed clinical features of all positive patients were noted. Representative enterovirus-positive samples were sequenced for viral; protein 1 (VP1) gene for type identification. The sequences were analysed by the Basic Local Alignment Search Tool (BLAST) software and enterovirus genotyping tool v1.0. A maximum likelihood tree was constructed using MEGA11.

Results

Out of 133 samples tested, 75.1% (n = 100) samples were positive for enterovirus by real-time PCR. The average duration of onset was 16.9 hours. Subconjunctival haemorrhage was observed in 67% of the patients, while 30% of cases had conjunctival chemosis. The causative agent of the current outbreak was identified as Coxsackievirus A24v G-IV. A sequence homology of 96–99% and pairwise distance divergence of 2.9% was observed between study sequences and recent sequences of China outbreak. Mutation analysis showed four common mutations (T281C, A311G, T332C, and T395C) with those of Chinese sequences, however no new mutations were identified in current study sequences, suggesting a close relatedness to CVA24 sequences from China.

Conclusions

Coxsackievirus A24v continues to be an important agent of Acute Haemorrhagic conjunctivitis in North India. In view of its highly contagious nature, effective and timely public health measures are required to limit its spread.

Haemorrhagic Conjunctivitis

Coxsackievirus

Outbreak

Viral

Acute Haemorrhagic Conjunctivitis (AHC) is a highly contagious viral conjunctivitis characterized by sudden onset of symptoms which include redness, ocular discomfort and watering. It is primarily caused by enteroviruses including Coxsackievirus A24v (CVA24v) and Enterovirus 70 (EV70) and to a lesser extent by Adenovirus 11.^[1] Initial outbreak of this entity was first reported in 1969 in a suburb of Accra, Ghana.^[2],[3] Since then, the disease has spread to many parts of the world, causing localized outbreaks, affecting millions of people, and has become a major global public health problem.

EV70 and CVA24v, the common causative agents belong to the family Picornaviridae and are non-enveloped positive sense single stranded RNA viruses.^[4] There are no known differences in signs and symptoms between the two etiological agents with the common symptoms being ocular pain, redness, swelling of the eyelids, irritation and eye discharge. The diagnostic sign of acute haemorrhagic conjunctivitis is the presence of small petechial haemorrhages mostly noticed in the inferior fornix along with an intense follicular reaction. The disease usually has a self-limiting course and is treated only symptomatically. Although rare, corneal involvement as well as neurological complications have been reported in a few studies.^[5],[6],[7]

EV70 and CVA24v related isolated outbreaks have also been reported previously from India.^{[8],[9],[10],[11]} Its incidence is more during hot and humid weather (August and September). The present paper reports an outbreak of AHC that occurred in Chandigarh, North India during the rainy season in the months of July-August 2023.

Patient enrolment

A trained medical personnel collected the conjunctival swab samples from patients with suspected viral conjunctivitis presenting to the Advanced Eye Center (AEC) of the Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh between July 24th and 15th August 2023. Around 500 patients presented with red eyes within 4 weeks. All patients with clinical signs and symptoms suggestive of acute red-eye, presenting to the clinic within 36 hours of the onset of symptoms, and providing written informed consent were included in the study.

Sample and data collection

The clinical details of the patients were recorded on a proforma. All enrolled patients were subjected to a detailed ophthalmic examination including best-corrected visual acuity (BCVA) as well as slit lamp biomicroscopic examination. Since the samples were collected during an outbreak situation with the need for early diagnosis, the ethics clearance was taken retrospectively from the Institute Ethics Committee and the proposal was cleared vide letter number IEC-INT/2023/STUDY-1430 dated 26.12.2023.

A trained medical personnel collected the conjunctival swab samples following all aseptic precautions. In the case of bilateral involvement, the eye that reported symptoms for a shorter duration was chosen to increase the chances of virus detection. The samples were transported to the Department of Virology in 3ml viral transport media (VTM) in a cold chain (ice packs). The samples were vortexed, swabs discarded, and samples stored in aliquots at -80^oC until tested.

Nucleic acid extraction and PCR amplification

The total nucleic acid was extracted using the automated extraction platform NucliSENS® easyMAG® system (BioMerieux, Inc, USA) with an initial sample volume of 200µl. The final elution volume was set to 60µl and the extracted nucleic acid was used for enterovirus testing by one-step real-time reverse transcriptase-polymerase chain reaction (RT-PCR) targeting the 5’UTR gene using previously published primers and probes^[12]. All samples with a cycle threshold value (Ct value) of ≤ 34, and clearly observable sigmoid graph on real time RT-PCR run were considered positive. The enterovirus negative samples were tested for adenovirus DNA by conventional PCR targeting the hexon gene as described earlier.^[13]

Enterovirus typing

For enterovirus typing, representative samples with cycle threshold value (Ct value) ≤ 25 were selected and subjected to sequencing of the VP1 region using a semi nested PCR approach as per WHO enterovirus surveillance guidelines,^[14] with slight modifications, using primer sets described by Nix et al.^[15] Briefly, for cycle 1, 10µl of cDNA was synthesized (Applied Biosystems) and added to the PCR master mix containing 10µm primer each of SO224 (Forward primer) and SO222 (reverse primer), 20mM dNTPs, 10X Taq buffer A (with 20mM MgCl₂) and Taq Polymerase enzyme (5U/µl) (BR Biochem Life Science Pvt Ltd, New Delhi) to a final volume of 50µl. The cycling conditions used were 94^oC for 12 min, followed by 40 reactions of denaturation at 95^oC for 30 sec, primer annealing at 42^oC for 30 sec and extension at 60^oC for 45 sec with a final extension step at 72^oC for 5 min. The second round of semi-nested PCR was performed using primer sets AN88 and AN89 with 3µl product from the first cycle as template in the 2nd cycle of nested PCR making 50 µl reaction. The PCR cycling parameters were the same as in the first round except that the annealing temperature was set at 50^oC for 30 seconds. The Coxsackievirus A16 strain was used as positive control in the PCR. The PCR products were run on 2% agarose gel for the presence of desired product of approximately 740bp and 370 bp for first and 2nd round of semi nested PCR cycles respectively.

Sanger sequencing and results analysis

The PCR positive samples having adequate concentration of viral RNA and showing prominent bands on agarose gel electrophoresis post conventional PCR were taken forward for sequencing. The gel purification was performed using the RBC HiYeld ^TM Gel/PCR DNA extraction (QDF 100, Real Biotech Corporation, Taiwan) kit using manufacturer's instructions. These products were sequenced using the Sanger method. The sequences obtained were analysed, trimmed and edited using GeneStudio, Bioedit. The nucleotide sequences thus generated were used to identify the enterovirus genotype using NCBI BLAST and enterovirus genotyping tool (https://www.rivm.nl/mpf/typingtool/enterovirus/). The sequences were aligned using MEGA 11 (64 bit) software and a phylogenetic tree was constructed using the maximum likelihood method with 1,000 bootstraps.

Around 500 patients with complaints of red eye and other symptoms of AHC presented to OPD during the outbreak. Based on the inclusion criteria, 133 subjects from all age groups were recruited between July 24th to August 15th, and 100 (75.1%) were positive for enterovirus by real time RT-PCR. The Ct values in positive samples ranged between 20 to 34. Among them, 25 samples exhibited Ct values ranging from 20 to 24, 45 had Ct values between 25 and 29, and 30 had Ct values more than 30. The 33 enterovirus negative subjects were subjected to testing for adenovirus by conventional PCR and none was found to be positive.

Demographic details

The age of the positive enterovirus positive patients ranged from 2–65 years with the maximum positivity being reported in the age group of 30–40 years (26%), followed by 20–30 years (18%). Paediatric subjects (< 18 years) constituted 31% of the positive cases while 3% of the positive subjects were > 60 years of age .The male: female ratio was 1:9. The average duration of onset was 16.9 hours with 53% of the patients having bilateral involvement. A prodrome of fever was present in 21% of the positive patients while 57% patients gave a history of contact with a patient of “red eye”.

Signs and symptoms:

All the patients presented with varying degrees of redness along with follicular reaction. (Fig. 1A,B,C) The associated symptoms included pain, foreign body sensation, burning sensation, itching and mild mucopurulent discharge. Bilateral ocular involvement was seen in 53% subjects. None of the patients developed corneal involvement. The severity of signs and symptoms have been depicted in Table 1.

The subconjunctival haemorrhage was observed in 67 (67%) patients. The degree of haemorrhage was graded as mild, moderate, severe and very Severe on slit lamp biomicroscopic examination. The degree was graded as mild (Grade 1: One quadrant of palpebral conjunctiva/petechial haemorrhage anywhere in the bulbar conjunctiva/palpebral conjunctiva) in 48%; moderate (Grade 2: Two quadrants of bulbar conjunctiva) in 9%,; severe (Grade 3: Three quadrants of bulbar) in 6% (Fig. 1D) and very severe (Grade 4: all four quadrants of bulbar conjunctiva) in 4% of the patients.

Nearly 21% (21/100) of patients reported history of medication use in the eye prior to presenting in the cornea clinic out of which 13% had history of instillation of antibiotic alone, 4% patients had instilled antibiotic steroid combination drops, while 4% patients had instilled tear substitutes alone.

Phylogenetic analysis:

In order to identify the particular kind of enterovirus, eight representative samples were sequenced for VP1 gene by Sanger method. The sanger sequencing results with clear chromatograms were analysed using NCBI BLAST, All of the sequences displayed over 98% identity to the Enterovirus C, i.e. Human Coxsackievirus A24 (CV-A24), particularly the CVA24v. Consequently, the etiological agent for the outbreak was found to be CVA24v (sub genogroup IV). The sequences were submitted to NCBI gene-bank with accession numbers OR526421-OR526423 and OR795627-OR795631. The phylogenetic analysis was done for study sequences and sequences from previously reported AHC outbreaks. The Neighbour-joining tree was constructed with 1000 bootstrap replicates. The phylogenetic analysis grouped the eight sequences from the outbreak from North-India along with the Coxsackievirus A24 (OR352715, OR352718) strain collected in a similar outbreak from China during 2023 (Fig. 2). The Pairwise distance analysis revealed that Coxsackievirus A24 (OR352715) strain had a pair-wise distance divergence of 2.9% from the outbreak strains of current study based on the partial VP1 region. Additionally, the study sequences showed 98% similarity with the outbreak sequences from those of Pakistan and Arunachal Pradesh, revealing the same etiological agent responsible for global outbreak. Alignment of the complete VP1 sequences of our strains and worldwide strains through Muscle software revealed the presence of four mutations namely the T281C, A311G, T332C, and T395C substitution common with the Chinese strains (OR352715, OR352718). We did not find any novel mutations in the strains from the current study.

Infection control measures:

There was a sudden surge in the number of patients presenting with red eye in the month of July 2023. When the number of patients reached a figure of 50 per day, strict infection control measures were initiated. Our tertiary eye care facility is a five level building with various speciality clinics on different floors. A separate “Red Eye Clinic” was set up on the ground floor with a designated resident doctor (trained ophthalmologist), a nursing staff for a slit lamp biomicroscopic examination, and a trained resident for collecting and transporting the swabs. Sign boards were displayed in bold letters at the entry of the centre for specified directions to the patient. Patients were asked to maintain social distancing and were examined under strict aseptic precautions. They were instructed to maintain personal and family hygiene. Standard of care treatment as for any viral conjunctivitis was given which included Hydroxy-propyl-methylcellulose, Chlorpheniramine, Naphazoline containing eye drops for symptomatic relief. Cold compresses and oral anti-inflammatory agents were added wherever required. The patients were asked to follow up if the symptoms persisted even after one week. All the experiments were carried out in a Biosafety level 2 laboratory with all safety precautions. On-site disinfection by autoclaving was done for the contaminated infectious waste (including the VTMs and pipette tips).

The present study reports an outbreak of acute haemorrhagic conjunctivitis which occurred in July-August 2023 in North India of patients presenting to our tertiary care hospital for treatment. At this time of the year, the AHC outbreaks were being reported from many of the South East Asian countries including Vietnam, Pakistan, Nepal and Ghana ^{[16],[17],[18]} and also from other parts of our country.

The AHC outbreak was reported in a tertiary care North Indian hospital wherein a sudden surge in the outpatients presenting with red eye was noticed. In the current study, and etiological agent was identified in more than 75% cases by universal pan-enterovirus primers^[19]. VP1 sequencing confirmed the causative agent as Coxsackievirus A24v from sub genogroup IV. The samples from the enterovirus PCR negative patients were tested for adenovirus and none was found to be positive. Reports of similar outbreak from Pakistan also showed that the agent responsible was Coxsackie A24v genotype IV.^[18]

The history of enteroviral conjunctivitis outbreaks can be traced back to 1969, when an unusual type of epidemic conjunctivitis was reported in Ghana with 8981 clinical cases being reported.^[2] Thereafter multiple isolated outbreaks have been reported from the different parts of the world including India. In the previous outbreaks, mixed aetiology in single outbreak i.e. EV70 and CVA24v have also been observed.^[20] The earliest evidence of EV70 infection in India came from Bombay (western India) in 1971-72,^[11] and the first isolation was reported from a single case during a small epidemic in southern India in 1975. Previous outbreaks in the North Indian region have been reported from New Delhi in the year 1981, 1986 and 1988.^{[9],[10],[11]} AHC due to EV70 appeared to have re-emerged in North India after more than three decades. During the intervening period, a Coxsackievirus A24v was circulating as a cause of AHC in this region of India. Outbreaks of AHC caused by CVA24v have also been reported from Gujarat and Maharashtra in the year 2003.^[20]

Most of the cases in our study occurred among adults 30–40 years of age (26%). In an earlier outbreak reported from Okinawa, Japan in the year 1994 ^[22], 62% of the subjects were in the age group of 11–15 years. In another outbreak reported from Maharashtra, India, in 2007 ^[21]; most of the patients were young adults in the 21–30 years of age group. A male predominance was observed in our study. Zhang et al have also reported 1.4 fold higher incidence of AHC in male subjects as compared to female subjects in China.^[23]

The cases peaked in the month of July and then declined by the end of August. Distinct cyclical and seasonal patterns of AHC outbreaks have been observed in most Southeast Asian countries with the cases peaking during the rainy season every 2–3 years. The high temperature and humidity at this time of the year may possibly be facilitating the rapid spread of CVA24v strains during this part of the year.^[23]

In this study, representative 8 samples sequenced for VP1 gene showed 96–99% identity with strains from recent outbreak in China (June, 2023) whereas 91–93% identity was seen with strains previously circulating in India. Mixed outbreaks of adenovirus and CVA24v have recently been reported from India in the year 2022^[24]. The earlier report of this outbreak reported genotyping using 5’UTR region ^{[25, 26]} In the current study, the genotype confirmation was done using the WHO described protocol for VP1 typing which revealed the presence of four mutations namely the T281C, A311G, T332C, and T395C substitution common with the Chinese strains (OR352715, OR352718). As very few sequences for VP1 are available from India, we could not find the transmission patterns. Also, we did not find these mutations in previously submitted sequences worldwide, therefore, a close monitoring in the form of regular surveillance is required to further confirm the role of such substitution mutations.

Signs and symptoms of acute haemorrhagic conjunctivitis have been described in limited studies. In a Japanese study, conjunctival hyperaemia was present in all patients, and subconjunctival haemorrhage, superficial punctate keratitis and preauricular lymphadenopathy were present in 24.0%, 11.7% and 9.3% of AHC cases, respectively.^[27] In 1996 in a study conducted at AIIMS, New Delhi, 13 patients with AHC caused by Enterovirus 70 were seen and all had bilateral ocular involvement with redness and watering of the eyes, mild photophobia, and severe foreign body sensation.^[5] In their small cohort of patients, superficial punctate keratitis was noted along with varying degrees of subconjunctival haemorrhage. In our study, subconjunctival haemorrhage was present in 62% of the patients, while none of the patients had corneal involvement or lymphadenopathy. Most of the patients had sudden onset of redness, watering and mild mucopurulent discharge. A characteristic feature of this conjunctivitis was that patients could clearly discern the exact time of onset of redness which was within hours (average 16 hours) in all the patients with nearly 57% patients having bilateral involvement. This is precisely the first study to describe the detailed signs and symptoms of 100 proven enteroviral conjunctivitis cases caused by CVA24v. The key clinical features observed in AHC are distinct from cases of adenovirus conjunctivitis which is the most common cause of viral conjunctivitis (70% of the conjunctivitis worldwide).^[28],[29] First and foremost is the sudden onset of redness with mild mucopurulent discharge with simultaneous involvement of the other eye or the other eye getting involved within few hours of onset of the previous eye; this bilateral involvement was seen in 53% of our patients. Secondly, the rapid onset of disease within hours which is so sudden that our patients were able to recall the exact time of the day when the symptoms started; the average duration of onset of disease was 16.9 hours by the time the patients reported at our centre. This actually corroborates with the incubation period of the enterovirus which in literature has been reported to be 1 day.^[30] This is unlike adenoviral disease which presents as a biphasic illness beginning with an infective phase where the symptoms peak at the 3rd to 4th day followed by an inflammatory phase, which tends to begin 7–10 days after the initial infection as the virus shedding continues.^[28] Another diagnostic clinch is the presence of petechial haemorrhages in the inferior fornix or frank subconjunctival haemorrhage in most of the patients on detailed slit lamp examination. These were seen in majority of our patients at presentation (64%) in the present study; unlike in patients of adenoviral conjunctivitis (exact incidence is not known). The incidence may as actually be close to 100% in this cohort of population of viral conjunctivitis, however due to intense hyperaemia in the acute phase the haemorrhages may not be appreciated and may be visible only during the natural course of the disease once the superficial congestion fades.

Enteroviral conjunctivitis is a highly contagious ocular condition, mainly transmitted through hand-to-eye-to-hand contact, but is self -limiting. Coxsackievirus A24v is a small non-enveloped virus which is highly resistant to most of the disinfectants used in day-to-day practice. The most commonly used sanitizer containing 70% alcohol is also not effective to kill this virus. In addition to its impervious nature, it has an extremely short incubation period, thus spreading rapidly, attaining huge numbers in a few days. Timely effective interventions were carried out to control this outbreak in our facility. The patients were educated to avoid hand to eye contact, frequent handwashing and avoid sharing towels, beddings and other items of personal use with other family members. The staff members from the institute who developed AHC were asked to refrain from work for one week as ours is a tertiary care clinical set up and patients from several states in North India are referred for various ailments. Moreover, disinfectants and sanitizers containing quaternary ammonium compounds were advocated. They include chlorhexidine, benzalkonium chloride, didecyldimethylammonium chloride and alkyl dimethyl benzyl ammonium saccharinate.^[31] These compounds mostly inactivate viruses by solvating and disrupting lipid envelopes.

The present study reports the molecular and clinical profile of acute haemorrhagic conjunctivitis outbreak and its control measures in a tertiary eye care centre in North India. The VP1 region analysis suggests four substitution mutations from previously reported CVA24v. The close relationship with 2023 China isolates, necessitates the need for effective surveillance and public health measures to curtail the spread of AHC across various countries. It is important to have continuous functional surveillance systems for early diagnosis and prompt control of such outbreaks. The current study did have a downsides though, in that it only included individuals who presented to the institute for testing; as a result, the real number of patients may have been higher. The instances that were actually collected may just represent the tip of the clinical iceberg.

Financial support

Deepanjli Sharma (File no: 09/0141(15439)/2022-EMR-I) gratefully acknowledges CSIR for awarding the Research Fellowship (JRF).

Author Contribution statement

The manuscript has been read and approved by all the authors, that the requirements for authorship have been met, and that each author believes that the manuscript represents honest work. Dr. Anchal and Dr. Deepanjali have contributed in the basic preparation of the manuscript. All authors have contributed in the management of the outbreak as well as writing of the manuscript.

Conflict of interest statement:

There is no conflict of interest for any author.

Author Contribution

A.T and D.S wrote the main manuscript. M.S and A.G guided in the management of this outbreak. A.S, P.C, V.A , A.G contributed in the molecular characterisation of the coxsackie virus. C.M and K.G helped in the Final preparation of the draft.

Acknowledgements:

The authors thank the Virology and the Ophthalmology Department to carry out this study

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Table 1: Table describing symptoms and signs of Coxsackie A-24v conjunctivitis.

Symptoms

	None	Mild	Moderate	Severe
Pain^a	18(18%)	65(65%)	17(17%)	0
Foreign Body sensation	14(14%)	56(56%)	28(28%)	2(2%)
Itching	65(65%)	28(28%)	6(6%)	1(1%)
Signs
Lid Oedema	36(36%)	45(45%)	18(18%)	1(1%)
Chemosis^b	70(70%)	28(28%)	1(1%)	1(1%)
Discharge^c	30(30%)	51(51%)	19(19%)	0
Follicles^d	0	55(55%)	42(42%)	3(3%)

a. Pain: None, mild: 1-3 (Mild Pain (nagging, annoying, interfering little with ADLs {Activity of daily life, Moderate, 4-6 (interferes significantly with ADLs) , Severe Pain: 7-10 (disabling; unable to perform ADLs)

b. Conjunctival oedema: None, Slight (oedema in one segment of bulbar conjunctiva), Moderate (uniform and diffuse oedema throughout bulbar Conjunctiva), Severe conjunctival chemosis with profusion of conjunctiva outside of palpebral slit)

c. Secretions: none, mild: only with slit lamp, moderate: secretions visible without slit lamp, severe: abundant secretions which can produce adherence of both eyelids

d. Palpebral conjunctiva: Slight (localised formation of follicles/papillary/mixed under 1mm diameter), Moderate (diffuse formation of follicles / papillary/mixed under 1mm diameter), Severe (abundant formation of follicles / papillary/mixed over 1 mm diameter)

No competing interests reported.

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Clinical and molecular investigation of Acute Haemorrhagic Conjunctivitis outbreak in North India

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