Outbreak of Adenovirus serotype 8 keratoconjunctivitis in Puducherry , South India : a molecular epidemiological study

Dear Editor, The present study reports an outbreak of conjunctivitis due to human adenovirus serotype 8 (HAdV-8) for the first time from India. HAdV-8, was detected in 16 samples by Multiplex PCR. Eleven strains were sequenced, analysed by phylogenetic tree with pre-epidemic and global isolates and revealed no variation with pre-epidemic strains. Acute viral conjunctivitis is the most common cause of infectious conjunctivitis. It can be caused by several viruses such as Human adenoviruses (HAdV), Herpes virus, Enterovirus 70 and Coxsackie virus A24 [1]. Adenovirus is the most common agent of viral conjunctivitis representing 15%-70% of all cases of conjunctivitis worldwide [2]. HAdV (Adenoviridae family) is divided into 7 species (A to G) and 68 serotypes [3]. Serotypes 3, 4, 7, 8, 19 and 37 are responsible for 89% of the cases of viral conjunctivitis worldwide [4]. Adenoviruses are non-enveloped and show resistance to both physical and chemical agents of disinfection. Their stability against such agents make them more infectious and facilitates their transmission in the community through fomites, ophthalmic instruments and even through ocular drops [5]. Information on the monthly incidence of adenoviral conjunctivitis is essential to prevent further infection [6]. In the month of October and November 2014, an outbreak of acute viral conjunctivitis occurred in Puducherry, India, during the rainy season. In this context, the aim of the present study was: to detect adenovirus by (i) viral culture, (ii) immunofluorescence assay (IFA) and (iii) multiplex polymerase chain reaction (PCR). We also conducted a molecular epidemiological study on adenovirus and its serotypes.

Dear Editor, The present study reports an outbreak of conjunctivitis due to human adenovirus serotype 8 (HAdV-8) for the first time from India.HAdV-8, was detected in 16 samples by Multiplex PCR.Eleven strains were sequenced, analysed by phylogenetic tree with pre-epidemic and global isolates and revealed no variation with pre-epidemic strains.
Acute viral conjunctivitis is the most common cause of infectious conjunctivitis.It can be caused by several viruses such as Human adenoviruses (HAdV), Herpes virus, Enterovirus 70 and Coxsackie virus A24 [1].Adenovirus is the most common agent of viral conjunctivitis representing 15%-70% of all cases of conjunctivitis worldwide [2].HAdV (Adenoviridae family) is divided into 7 species (A to G) and 68 serotypes [3].Serotypes 3, 4, 7, 8, 19 and 37 are responsible for 89% of the cases of viral conjunctivitis worldwide [4].Adenoviruses are non-enveloped and show resistance to both physical and chemical agents of disinfection.Their stability against such agents make them more infectious and facilitates their transmission in the community through fomites, ophthalmic instruments and even through ocular drops [5].
Information on the monthly incidence of adenoviral conjunctivitis is essential to prevent further infection [6].In the month of October and November 2014, an outbreak of acute viral conjunctivitis occurred in Puducherry, India, during the rainy season.In this context, the aim of the present study was: to detect adenovirus by (i) viral culture, (ii) immunofluorescence assay (IFA) and (iii) multiplex polymerase chain reaction (PCR).We also conducted a molecular epidemiological study on adenovirus and its serotypes.

The study
An outbreak of acute viral conjunctivitis was observed during routine surveillance in the months of October and November 2014.A total of 42 patients (24 males and 18 females) who presented with viral conjunctivitis in the outpatient department of Ophthalmology at the Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER) were included in the study.Dacron swabs were used to sample material from the inferior palpebral conjunctiva, transported in viral transport medium (VTM) and processed in the virology laboratory.Samples were vortexed and centrifuged at 10,000 rpm for 30 minutes at 4 o C. The supernatant was used for both viral culture and PCR.The pellet was used for IFA.Viral culture was carried out by inoculating 0.5 ml of supernatant into a 24 hours old Hep2 cell line and incubated at 37°C for 30 minutes in a CO2 incubator in order to achieve enhanced absorption.Maintenance medium (Dulbecco's minimal essential medium supplemented with 2% foetal calf serum) was added and incubated at 37°C.The culture flask was regularly observed for typical cytopathic effects of adenoviruses (grape like clusters).IFA was carried out on all positive cultures.If there were no cytopathic effects at the end of seventh day, blind passage was done and the sample was reported negative after another 7 days.IFA was also carried out for rapid identification of adenovirus directly from samples.The pellet, isolated during sample processing was spotted on a Teflon coated slide.Immunofluorescence staining was carried out using primary mouse origin anti-adenoviral (Ab) directed against a hexon protein (Catalog No: MAB8051) and secondary anti-mouse FITC labelled Ab of goat origin (Catalog No: CA92590) (Chemicon Millipore, Temecula, California, USA).Staining procedures were carried out with appropriate positive and negative controls, and examined under fluorescence microscope for apple green fluorescence (Supplementary Figures 1  and 2) [7].
In our present work, PCR was carried out to detect adenoviral serotypes.Viral DNA was extracted from the supernatant fluid using a commercial kit (QIAGEN, Hilden, Germany).Adenovirus Universal Semi-nested PCR based on Hexon target region (HELINI Biomolecules, Chennai, India) was carried out to detect adenoviral DNA.The internal controls were run concurrently for each set of experiments along with positive and negative controls.Samples that were positive by the Universal Semi-nested PCR, were subjected to a multiplex PCR for identification of adenoviral serotypes such as 3, 4, 8, 7, 19 and 37 (primer sequences listed in Table 1).

Results and discussion
Epidemic Keratoconjunctivitis is a severe contagious eye disease frequently associated with Human adenovirus Serotype 8 (HAdV-8) [8].HAdV-8 as an etiological agent of EKC was first reported by Jawetz and Hamma in the year 1959.Till date, HAdV-8 remains an important agent of EKC worldwide and outbreaks have been reported from several studies [9][10][11].Results of the present work depict that out of 42 patients, 23 were positive for adenovirus by Universal Semi-nested PCR, IFA or viral culture (Table 2).Multiplex PCR was carried out on 16 PCR-positive samples and 4 PCR negative, viral culture positive samples for serotype detection.All 20 were identified as adenovirus serotype 8. Remaining 3 samples were positive only by IFA and not by other methods.Sequencing was carried out for 11 strains and all were confirmed to belong to serotype 8 (Gen bank accession No. KR150657-667).Additionally, two pre-epidemic strains were sequenced to be serotype 8 (KR084326-27).The sequences were aligned using Clustal-W algorithm.All the sequenced serotypes including the pre-epidemic isolates were aligned for phylogenetic analysis.The analysis revealed that the pre-epidemic and epidemic strains showed no variations in sequence   based on the unweighted pair group method with arithmetic mean (UPGMA) method (Figure1).To the best of our knowledge, this is the first study from India, reporting an outbreak of conjunctivitis due to adenovirus serotype 8. Hence, we compared our strains with the global isolates and a phylogenetic tree was constructed (Figure 2).In the present work, phylogenetic tree analysis revealed that our strains belong to two clusters (A5, A6, A7, A8, A9, A13, A16, Hiroshima strain) and (A2, A3, A10, A11, A14, A15, Spain, Kawasaki, Austria, Korea, Hungary, Fukushima strains).In each year, HAdV-8 accounts for the highest numbers of EKC cases worldwide.Previous studies from India reported outbreaks of conjunctivitis due to serotypes 3, 4, 7 [12] and a variant strain closely related to 2 and 6 [13].Hence this is the first report of viral conjunctivitis due to HAdV-8 from India.Furthermore, we observed that all the patients presented with single eye involvement and within three days of symptoms.

Figure 1 .
Figure 1.Phylogenetic analysis of pre-epidemic and epidemic strains.

Figure 2 .
Figure 2. Phylogenetic analysis of outbreak strains with global strains.

Table 1 .
Primers used in this study

Table 2 .
Number of samples positive for Adenovirus by various methods